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Presented  by 
T.   G.  Burt,  B.  0. 


COLLEGE    OF    OSTEOPATHIC    PHYSICIANS 
AND  SURGEONS  •    LOS  ANGELES,  CALIFORNIA 


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UNIVERSITY  OF  CALIFORNIA 

CALIFORNIA  COLLEGE  OF  MEDICINE 

LIBRARY 

JlIM      81971 
IRVINE,'  CALIFORNIA  92664 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/clncldiagnsismanOOtoddiala 


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Explanation  of  Plate  I 

»-  Stained  with  Wright's  stain.  All  drawn  to  same  scale. 
I,  Normal  red  corpuscle  for  comparison;  2,  normoblasts,  one  with 
lobulated  nucleus;  3,  megaloblast  and  microblast.  The  megaloblast 
shows  a  considerable  degree  of  polychromatophilia;  4,  blood-plaques, 
one  lying  upon  a  red  corpuscle;  5,  lymphocytes,  large  and  small;  6, 
large  mononuclear  leukocyte;  7,  transitional  leukocyte;  8,  polymor- 
phonuclear neutrophilic  leukocytes;  9,  eosinophilic '  leukocytes,  one 
ruptured;  10,  basophilic  leukocyte;  11,  neutrophihc  myelocyte.  The 
granules  are  sometimes  less  numerous  and  less  distinct  than  here  shown; 
12,  eosinophilic  myelocytes;  13,  basophilic  myelocyte;  14,  "irritation" 
or  "stimulation"  form,  with  small  vacuoles;  15,  degenerated  leukocytes: 
two  polymorphonuclear  neutrophiles,  one  ruptured,  one  swollen  and 
vacuolated;  and  a  "  basket  cell"  composed  of  an  irregular  meshwork 
of  nuclear  material;  16,  large  mononuclear  leukocyte  containing  pigment- 
granules;  from  a  case  of  tertian  malaria;  17,  four  stages  in  the  asexual 
cycle  of  the  tertian  malarial  parasite:  the  second  and  fourth  were  drawn 
from  the  same  slide  taken  from  a  case  of  double  tertian;  18,  red  corpuscle 
containing  tertian  parasite  and  showing  malarial  stippling;  19,  estivo- 
autumnal  malarial  parasites:  two  small  ring  forms  within  the  same 
red  cell,  and  a  crescent  with  remains  of  the  red  corpuscle  in  its  concavity. 


Clinical  Diagnosis 

A  MANUAL  OF  LABORATORY  METHODS 


BY 

JAMES    CAMPBELL    TODD.    Ph.  B..   M.   D. 

PROFESSOR  OF  CLINICAL  PATHOLOGY,  UNIVERSITY  OF  COLORADO 


Illustrated 


Fourth  Edition,  Revised  and  Reset 


PHILADELPHIA   AND  LONDON 

W.     B.     SAUNDERS     COMPANY 

1919 


)1H 


c 


Copyright,  1908,  by  W.  B.  Saunders  Company.    Revised,  reprinted,  and  recopy- 
righted  January,  igi2.     Reprinted  July,  1913.     Revised,  entirely  reset, 
reprinted,  and  recopyrighted  October,  1914.     Reprinted  Jan- 
uary, 1916,  and  September,  1917.     Revised,  entirely 
reset,  reprinted,  and  recopyrighted   June,   1918 


Copyright,  1918,  by  W.  B.  Saunders  Company 


Reprinted  February,  1919 


Reprinted  October,  1919 


PRINTED    IN    AMERICA 

PRESS    OF 

W.     B.     SAUNDERS     COMPANY 

PHILADELPHIA 


TO 

MY  FATHER 

3oe  M.  ^o^b,  fID.  S), 

THESE   PAGES  ARE 
AFFECTIONATELY   DEDICATED 


PREFACE  TO  THE  FOURTH  EDITION 

CJ 

In  the  present  edition,  as  in  the  preceding  one,  the 
scope  of  this  book  has  been  somewhat  extended  and  its 
size  increased.  It  is  hoped  that  its  value  has  thereby 
been  enhanced  without  sacrifice  of  the  simpHcity  and 
conciseness  which  were  its  original  aim.  As  before, 
chief  emphasis  has  been  laid  upon  methods  and  micro- 
scopic morphology. 

Much  of  the  new  material  is  the  outgrowth  of  ques- 
tions which  have  arisen  in  class-room  and  laboratory. 
To  one  who  sees  a  great  deal  of  the  work  of  students  in 
the  clinical  laboratory,  it  soon  becomes  evident  that 
errors  in  microscopic  diagnosis  spring  much  less  fre- 
quently from  ignorance  of  the  typical  appearance  of 
microscopic  structures  than  from  imperfect  preparation 
of  the  material,  faulty  manipulation  of  the  microscope, 
or  failure  to  recognize  extraneous  structures,  artifacts, 
and  other  misleading  appearances.  Such  sources  of 
error  have  been  given  especial  attention. 

In  order  to  keep  the  size  of  the  volume  within  bounds, 
room  has  been  made  for  the  new  matter  by  omissions 
and  condensations  so  far  as  these  have  seemed  wise, 
but  great  care  has  been  exercised  to  avoid  omission  of 
essential  ^details.  It  has,  in  fact,  been  found  necessary 
to  elaborate  many  subjects  which  seemed  to  have  been 
too  briefly  stated  in  past  editions  for  clear  understand- 
ing. Very  brief  descriptions  of  methods  and  micro- 
scopic structures  are  attractive,  but  only  too  often  they 

21396  ' 


8  PREFACE    TO    THE    FOURTH    EDITION 

are  worse  than  useless.  They  necessarily  omit  details 
which  seem  unimportant  in  themselves,  but  which  in 
reality  are  essential  to  guard  the  reader  against  errors. 
They  give  him  an  unfounded  confidence  which  is 
doomed  to  disillusionment  when  he  actually  attempts 
the  work  in  the  laboratory. 

The  changes  and  additions  are  widely  scattered 
throughout  the  book,  hence  most  of  them  evade  special 
mention.  The  use  of  colorimeters  and  of  the  pocket 
spectroscope  and  methods  of  matching  blood  for  trans- 
fusion have  been  given  at  some  length.  There  have 
also  been  included  sections  dealing  with  the  new  Bass 
and  Johns  concentration  method  for  malarial  parasites; 
the  fractional  method  of  gastric  analysis;  vital  staining 
of  blood-corpuscles;  resistance  of  red  corpuscles;  the 
mastic  reaction  in  the  spinal  fluid ;  the  Wilber  and  Addis 
method  for  urobilin  as  an  aid  in  diagnosis  of  pernicious 
anemia;  and  estimation  of  amylase  in  urine  and  feces 
in  diagnosis  of  pancreatic  disease.  The  chapter  upon 
sero-diagnostic  methods  has  been  revised  by  Professor 
Whitman  from  whose  pen  it  originated. 

In  a  book  which  deals  largely  with  clinical  micros- 
copy, accurate  pictures  of  microscopic  structures  should 
play  a  large,  if  not  predominant,  part.  They  give  in- 
formation which  cannot  be  conveyed  in  any  other  way. 
For  this  reason  the  illustrations  have  been  carefully 
revised.  The  poorer  illustrations  of  previous  editions 
have  been  omitted  and  90  new  black  and  white  pictures 
have  been  added,  making  an  increase  of  57  in  the  total. 
The  majority  of  the  new  pictures  are  photomicrographs 
by  the  author.  Inadequate  as  is  the  photomicrograph 
in  some  fields,  its  superiority  in  clinical  microscopy  can- 


PREFACE    TO    THE    FOURTH    EDITION  9 

not  be  questioned.  Of  the  colored  plates,  four  are  new 
in  this  edition.  Figure  i  of  Plate  II  was  made  under 
the  direction  of  Dr.  Stella  M.  Gardner  of  Chicago. 
The  remainder  of  the  new  colored  pictures  were  painted 
under  the  author's  supervision.  All  were  drawn  with 
painstaking  accuracy  from  actual  specimens,  in  most 
cases  with  the  aid  of  photomicrographs. 

To  all  those  of  his  present  and  former  pupils  whose 
suggestive  questions  have  been  an  influence  in  shaping 
the  book,  the  author  is  duly  grateful;  and  in  particular 
he  takes  pleasure  in  acknowledging  his  indebtedness  to 
Robert  C.  Lewis,  Professor  of  Physiology  and  Biochem- 
istry in  the  University  of  Colorado,  for  helpful  sugges- 
tions concerning  the  chemical  examination  of  the  urine 
and  of  the  gastric  contents. 

J.  C.  T. 

Henry  S.  Denison  Research  Labora- 
tories, University  of  Colorado. 
Boulder,  Colorado. 


PREFACE 


This  book  aims  to  present  a  clear  and  concise  state- , 
ment  of  the  more  important  laboratory  methods  which 
have  clinical  value,  and  a  brief  guide  to  interpretation 
of  results.  It  is  designed  for  the  student  and  practi- 
tioner, not  for  the  trained  laboratory  worker.  It  had 
its  origin  some  years  ago  in  a  short  set  of  notes  which 
the  author  dictated  to  his  classes,  and  has  gradually 
grown  by  the  addition  each  year  of  such  matter  as  the 
year's  teaching  suggested.  The  eagerness  and  care  with 
which  the  students  and  some  practitioners  took  these 
notes  and  used  them  convinced  the  writer  of  the  need 
of  a  volume  of  this  scope. 

The  methods  offered  are  practical;  and  as  far  as 
possible  are  those  which  require  the  least  complicated 
apparatus  and  the  least  expenditure  of  time.  Simplicity 
has  been  considered  to  be  more  essential  than  absolute 
accuracy.  Although  in  many  places  the  reader  is  given 
the  choice  of  several  methods  to  the  same  end,  the 
author  believes  it  better  to  learn  one  method  well  than 
to  leam  several  only  partially. 

More  can  be  learned  from  a  good  picture  than  from 
any  description,  hence  especial  attention  has  been  given 
to  the  illustrations,  and  it  is  hoped  that  they  will  serve 
truly  to  illustrate.     Practically  all  the  microscopic  struc- 


12  PREFACE 

tures  mentioned,  all  apparatus  not  in  general  use,  and 
many  of  the  color  reactions  are  shown  in  the  picture's. 

Although  no  credit  is  given  in  the  text,  the  recent 
medical  periodicals  and  the  various  standard  works  have 
been  freely  consulted.  Among  authors  whose  writings 
have  been  especially  helpful  may  be  mentioned  v.  Jaksch, 
Boston,  Simon,  Wood,  Emerson,  Purdy,  Ogden,  Ewald, 
Ehrhch  and  Lazarus,  Da  Costa,  Cabot,  Osier,  Stengel, 
and  McFarland. 

The  author  wishes  hereby  to  express  his  indebtedness 
to  Dr.  J.  A.  Wilder,  Professor  of  Pathology  in  the  Den- 
ver and  Gross  College  of  Medicine,  for  aid  in  the  final 
revision  of  the  manuscript;  and  to  W.  D.  Engel,  Ph.D., 
Professor  of  Chemistry,  for  suggestions  in  regard  to  de- 
tection of  drugs  in  the  urine.  He  desires  to  acknowl- 
edge the  care  with  which  Mr.  Ira  D.  Cassidy  has  made 
the  original  drawings,  and  also  the  uniform  courtesy  of 
W.  B.  Saunders  Company  during  the  preparation  of 
the   book. 

J.  C.  T. 

Denver,  Colorado. 


CONTENTS 


INTRODUCTION 

Page 

Use  of  the  Microscope 17 

CHAPTER  I 

The  Sputum 56 

Physical  Examination 59 

Microscopic  Examination 62 

Unstained  Sputum 63 

Stained  Sputum 74 

Chemic  Examination ;    .    .  94 

Sputum  in  Disease 95 

CHAPTER  II 

The  Urine 99 

Physical  Examination 102 

Chemic  Examination 116 

Normal  Constituents 123 

Abnormal  Constituents 149 

Microscopic  Examination 198 

Unorganized  Sediments 201 

Organized  Sediments 215 

Extraneous  Structures 239 

The  Urine  in  Disease 242 

CHAPTER  III 

The  Blood 249 

Methods  of  Obtaining .  252 

13 


14  CONTENTS 

Page 

Coagulation      257 

Hemoglobin 260 

Enumeration  of  Erythrocytes 270 

Color  Index      284 

Volume  Index 285 

Enumeration  of  Leukocytes 287 

Decrease  in  Number  of  Leukocytes 287 

Increase  in  Number  of  Leukocytes  ....'....    287 

Leukocytosis 288 

Leukemia      294 

Method  of  Counting  Leukocytes      294 

Enumeration  of  Blood-plaques 300 

Study  of  Stained  Blood      302 

Making  and  Staining  Blood-films 302 

Study  of  Stained  Films 314 

Blood  Parasites 345 

Bacteria 345 

Animal  Parasites 348 

Tests  for  Recognition  of  Blood 364 

Less  Frequently  Used  Methods 372 

Special  Blood  Pathology 380 

Anemia .    380 

Leukemia      387 

CHAPTER  IV 

The  Stomach 392 

Examination  of  the  Gastric  Contents 392 

Obtaining  the  Contents 393 

Physical  Examination 398 

Chemic  Examination      400 

Microscopic  Examination 414 

The  Gastric  Contents  in  Disease 417 

Additional  Examinations  which  Give  Information  as  to  the 

Condition  of  the  Stomach 4^9 

CHAPTER  V 

The  Feces 423 

Macroscopic  Examination 424 

Chemic  Examination      429 


CONTENTS  15 

Page 

Microscopic  Examination 436 

Functional  Tests 444 

CHAPTER  VI 

Animal  Parasites 448 

Protozoa 451 

Sarcodina 453 

Mastigophora  (Flagellata) 460 

Sporozoa 470 

Infusoria 471 

-    Platyhelminthes 472 

Nemathelminthes 492 

Arthropoda 511 

CHAPTER  VII 

Miscellaneous  Examinations         515 

Piis 515 

Peritoneal,  Pleural,  and  Pericardial  Fluids 520 

Cerebrospinal  Fluid 524 

Animal  Inoculation 534 

The  Mouth 535 

The  Eye 540 

The  Ear 543 

Parasitic  Diseases  of  the  Skin       543 

Milk 544 

Syphilitic  Material      548 

Semen 553 

Diagnosis  of  Rabies 555 

CHAPTER  VIII 

Bacteriologic  Methods 558 

Apparatus 558 

Sterilization      .' 562 

Preparation  of  Culture-tubes 563 

Culture-media 564 

Staining  Methods 571 

Methods  of  Studying  Bacteria 576 

Characteristics  of  Special  Bacteria 580 


l6  CONTENTS 

CHAPTER  IX 

Paoe 

Preparation  and  Use  of  Vaccines 585 

Preparation  of  Vaccine 585 

Method  of  Use 591 

Dosage 592 

Therapeutic  Indications 592 

Prophylactic  Use  of  Vaccines 594 

Tuberculins 594 

Tuberculin  in  Diagnosis 596 

Cutaneous  Test  for  Syphilis      598 

Schick  Test  for  Immunity  to  Diphtheria 599 

CHAPTER  X 

Serodiagnostic  Methods 600 

Immunity 600 

Apparatus .".... 603 

Reactions  Based  Upon  Immune  Bodies  of  the  Second 

Order 604 

The  Widal  Reaction 604 

Biologic  Identification  of  Unknown  Proteins.    .    .    .611 

Opsonins 615 

Reactions  Based   Upon  Immune   Bodies  of   the    Third 

Order 618 

Complement  Deviation  Test  for  Syphilis 619 

Complement  Deviation  Test  for  Gonorrhea       .    .    .  632 

Complement  Deviation  Test  for  Tuberculosis   .    .    .  633 

Cobra-venom  Test  for  Syphilis 636 

Appendix 639 

Staining  Solution 639 

Office  Laboratory  Equipment 643 

Weights,  Measures,  etc.,  with  Equivalents 653 

Temperature 654 

Index 655 


CLINICAL  DIAGNOSIS 


INTRODUCTION 
USE  OF  THE  MICROSCOPE 


There  is  probably  no 
laboratory  instrument 
whose  usefulness  de- 
pends so  much  upon 
proper  manipulation  as 
the  microscope,  and 
none  is  so  frequently 
misused  by  beginners. 
Some  suggestions  as  to 
its  proper  use  are,  there- 
fore, given  at  this  place. 
It  is  presumed  that  the 
reader  is  already  famil- 
iar with  its  general  con- 
struction (Fig.  i). 

For  those  who  wish 
to  understand  the  prin- 
ciples of  the  microscope 
and  its  manipulation — 
and  best  results  are  im- 
possible without  such  an 
understanding— a  care- 
ful study  of  some  stand- 
ard work  upon  micros- 
copy, such   as  those  of 

2 


Fig.  I. — Handle-arm  m\croscope: 
E,  Eye-piece;  D,  draw-tube;  T,  body- 
tube;  RN,  revolving  nose-piece;  O, 
objective;  PH,  pinion  head  for  coarse 
focusing;  MH,  micrometer  head  for 
fine  focusing;  HA,  handle-arm;  SS, 
substage;  S,  stage;  M,  mirror;  B, 
base;  R,  rack;  P,  pillar;  I,  inclination 
joint. 

17 


I 6  INTRODUCTION 

Carpenter,  Spitta,  and  Sir  A.  E,  Wright,  is  earnestly 
recommended.  It  is  also  recommended  that  the  be- 
ginner provide  himself  with  some  slides  of  diatoms,  for 
example,  Pleurosigma  angulatum,  Surirella  gemma,  and 
Amphipleura  pellucida,  costing  fifty  cents  each,  and 
with  some  good  preparations  of  stained  and  unstained 
blood.  The  blood  slides  can  easily  be  made  from 
one's  own  blood,  as  described  in  Chapter  III.  Faith- 
ful practice  upon  such  test-objects,  in  the  Hght  of  the 
principles  of  microscopy,  will  enable  the  student  to 
reach,  intelligently,  an  accuracy  in  manipulation  to 
which  the  ordinary  laboratory  worker  attains  only 
slowly  and  by  rule  of  thumb.  He  will  soon  find  that 
the  bringing  of  an  object  into  accurate  focus  is  by  no 
means  all  of  microscopy. 

Source  of  Light. — Good  work  cannot  be  done  with- 
out proper  illumination  and  this  is  therefore  the  first 
and  most  important  consideration  for  one  who  wishes 
to  use  the  microscope  effectively. 

The  light  which  is  generally  recommended  as  best  is 
that  from  a  white  cloud,  the  microscope  being  placed 
by  preference  at  a  north  window,  to  avoid  direct  sun- 
Hght.  At  any  other  window  a  white  window-shade  is 
desirable.  Such  light  is  satisfactory  for  all  ordinary 
work.  Artificial  light  is,  however,  imperative  for 
those  who  must  work  at  night,  and  is  a  great  conven- 
ience at  all  times.  Properly  regulated  artificial  light, 
moreover,  offers  decided  advantages  over  daylight 
for  critical  work.  Almost  any  strong  light  which  is 
diffused  through  a  frosted  globe  will  give  fair  results. 
The  inverted  Welsbach  light  with  such  a  globe  is 
excellent   as   is    also    the    Mazda   incandescent  lamp 


USE   OF  THE   MICROSCOPE 


19 


with  frosted  bulb.  Such  a  bulb  may  conveniently  be 
inclosed  within  a  tin  or  paste-board  box  with  small 
openings  in  the  back  for  ventilation  and  a  circular 
window  in  the  front  to  transmit  the  light.  At  the 
University  of  Colorado,  where  the  students  do  most  of 
their  microscopic  work  by  artificial  light,  the  lamp  shown 
in  Fig.  2  is  very  popular.     It  has  the  advantage  that  the 


-^ 


Fig.   2. — A  convenient  lamp  for  use  with  the  microscope. 


eyes  are  shaded  from  the  glare,  while  at  the  same  time 
there  is  abundant  light  for  drawing  or  writing  upon  the 
table  beside  the  microscope.  Its  cost,  with  Mazda  bulb, 
is  $1 .25.  All  such  lights  have  a  yellow  tinge,  to  counter- 
act which  a  blue  glass  disk,  usually  supplied  with  the 
microscope,  is  placed  in  a  supporting  ring  beneath  the 
condenser.     Recently   a   blue   glass   "daylight"   bulb 


20 


INTRODUCTION 


has  been  put  upon  the  market.  The  following  plan 
is  much  used  abroad,  and  gives  results  equal  to  the  best 
daylight:  A  Welsbach  lamp  or  strong  electric  light  is 
used,  and  a  spheric  glass  globe — a  6-inch  round-bottom 
flask  answers  admirably — is  placed  between  it  and  the 
microscope,  to  act  as  a  condenser  (Fig.  3).  The  flask 
should  be  at  a  distance  equal  to  its  diameter  from  both 
the  light  and  the  mirror  of  the  microscope.     In  order 


Pig.  3. — Illumination  with  water-bottle  condenser. 


to  filter  out  the  yellow  rays  the  flask  is  filled  with  water 
to  which  have  been  added  a  few  crystals  of  copper  sul- 
phate and  a  little  ammonia. 

Within  the  past  few  years  manufacturers  have  paid 
more  attention  than  formerly  to  means  of  artificial 
illumination  and  most  of  them  now  ofi^er  several  types 
of  lamp.  Two  good  tj-pes  are  shown  in  Figs.  4  and  5. 
Both  can  be  fitted  with  light  filters  made  of  the  newly- 
invented  ''daylight  glass,"  which,  when  used  with  the 


USE    OF    THE   MICROSCOPE  21 

nitrogen-filled  tungsten  lamp,  transmits  a  light  prac- 
tically indistinguishable  from  daylight  either  visually 
or  spectrophotometrically. 

The  microscope  lamp  should  not  stand  at  so  great 


Fig.  4. — Small  microscope  lamp  with  daylight-glass  filter. 


Fig.  5. — An  excellent  type  of  microscope  lamp  suitable  both  for  ordi- 
nary work  and  for  dark-ground  illumination. 

a  distance  from  the  microscope  that  its  image  fails  to 
fill  the  aperture  of  the  condenser — a  condition  which 
one  can  readily  detect  by  removing  the  ocular  and 
looking  down  the  tube. 


22  INTRODUCTION 

Forms  of  Illumination. — After  one  has  arranged  the 
microscope  in  proper  relation  to  the  source  of  hght, 
whether  this  be  daylight  or  any  of  the  artificial  sources 
mentioned  above,  the  next  problem  is  to  secure  an 
evenly  illuminated  field  of  view  without  mottling  or  any 
trace  of  shadows.  This  is  accomplished  by  manipu- 
lating the  mirror  and  the  condenser.  Following  this 
the  direction  and  the  amount  of  light  must  be  consid- 
ered in  relation  to  the  character  of  the  object  under 
examination. 

Illumination  may  be  either  central  or  oblique,  depend- 
ing upon  the  direction  in  which  the  light  enters  the 
microscope.  To  obtain  central  illumination,  the  mirror 
should  be  so  adjusted  that  the  light  from  the  source 
selected  is  reflected  directly  up  the  tube  of  the  micro- 
scope. This  is  easily  done  by  removing  the  eye-piece 
and  looking  down  the  tube  while  adjusting  the  mirror. 
The  eye-piece  is  then  replaced,  and  the  light  reduced  as 
much  as  desired  by  means  of  the  diaphragm. 

Oblique  illumination  is  obtained  in  the  more  simple 
instruments  by  swinging  the  mirror  to  one  side,  so  that 
the  light  enters  the  microscope  obliquely.  The  more 
complicated  instruments  obtain  it  by  means  of  a  rack 
and  pinion,  which  moves  the  diaphragm  laterally. 
Beginners  frequently  use  oblique  illumination  without 
recognizing  it,  and  are  thereby  much  confused.  If  the 
light  be  oblique,  an  object  in  the  center  of  the  field  will 
appear  to  sway  from  side  to  side  when  the  fine  adjust- 
ment is  turned  back  and  forth. 

The  amount  of  light  admitted  is  also  important.  It  is 
regulated  by  the  diaphragm. 

The  bulk  of  routine  work  is  done  with  central  illumi- 


USE    OF    THE    MICROSCOPE 


23 


nation,  and,  therefore,  every  examination  should  begin 
with  it.  Each  of  the  forms  of  illumination,  however — 
central  and  oblique,  subdued  and  strong — has  its  special 
uses  and  demands  some  consideration  here.  The  well- 
known  rule,  "Use  the  least  light  which  will  show  the 
object  well,"  is  good,  but  it  does  not  go  far  enough. 

In  studying  any  microscopic  structure  one  considers: 
(i)  its  color,  (2)  its  outline,  and  (3)  its  surface  contour. 
No  one  form  of  illumination  shows  all  of  these  to  the 


\..>  I 


Fig.  6. — a.  Hyaline  casts,  one  containing  renal  cells;  properly  sub- 
dued illumination;  b,  same  as  a;  strong  illumination.  The  casts  are 
lost  in  the  glare,  and  only  the  renal  cells  are  seen.  (From  Greene's 
"  Medical  Diagnosis. ") 


best  advantage.  It  may,  therefore,  be  necessary  to 
change  the  illumination  many  times  during  a  micro- 
scopic examination.  To  see  color  best,  use  central  illu- 
mination with  strong  light.  The  principle  is  that  by 
which  a  stained  glass  window  shows  the  purest  color 
when  the  light  is  streaming  through  it.  Strong  central 
light  is,  therefore,  to  be  used  for  structures  such  as 
stained  bacteria,  whose  recognition  depends  chiefly  upon 
their  color,  and,  alternating  with  other  forms,  for  stained 


24  INTRODUCTION 

structures  in  general.  To  study  the  outline  of  an  object 
use  very  subdued  central  illumination.  The  diaphragm 
is  closed  to  the  point  which  trial  shows  to  be  best  in  each 
case.  This  illumination  is  required  by  delicate  colorless 
objects,  such  as  hyaline  tube-casts  and  cholesterin 
crystals,  which  are  recognized  chiefly  by  their  outline. 
The  usual  mistake  of  beginners  is  to  work  with  the  dia- 
phragm too  wide  open.  Strong  light  will  often  render 
semitransparent  structures  entirely  invisible  (Fig.  6). 
To  study  surface  contour  use  oblique  light  oj  a  strength 
suited  to  the  color  or  opacity  oJ  the  object.  In  routine 
work  oblique  illumination  is  resorted  to  only  to  study 
more  fully  some  object  which  has  been  found  with  cen- 
tral illumination,  as,  for  instance,  to  demonstrate  the 
cylindric  shape  of  a  hyaline  tube-cast. 

Dark -ground  illumination  consists  in  blocking  out 
the  central  rays  of  light  and  directing  the  peripheral 
rays  against  the  microscopic  object  from  the  side.  Only 
those  rays  which  strike  the  object  and  are  reflected 
upward  pass  into  the  objective.  The  object  thus  ap- 
pears bright  upon  a  black  background.  By  means  of 
this  form  of  illumination  very  minute  structures  can  be 
seen,  just  as  particles  of  dust  in  the  atmosphere  become 
visible  when  a  ray  of  sunlight  enters  a  darkened  room. 

Dark-ground  illumination  for  low-power  work  can  be 
obtained  by  means  of  the  ring  stops  with  central  disks 
which  accompany  most  microscopes  when  purchased. 
The  stop  is  placed  in  a  special  ring  beneath  the  con- 
denser. When  the  regular  stop  is  not  at  hand,  one  can 
use  the  glass  disk  which  is  generally  supplied  with  the 
microscope  or  an  extra-large  round  cover-glass,  in  the 
center  of  which  is  pasted  a  circular  disk  of  black  paper. 


USE    OF    THE    MICROSCOPE  25 

The  size  of  the  black  disk  depends  upon  the  aperture  of 
the  objective  with  which  it  is  to  be  used,  and  can  be 
ascertained  by  trial. 

For  oil-immersion  work  a  special  condenser  is  neces- 
sary. This  is  sold  under  the  name  of  reflecting  conden- 
ser, "dunkelfeld,"  dark-field  illuminator,  etc.  With 
some  makes  it  is  placed  upon  the  stage  of  the  micro- 
scope; with  others  it  is  substituted  for  the  regular  con- 
denser. It  requires  an  intense  light,  like  that  given  by 
a  nitrogen-filled  tungsten  lamp  or  a  small  arc-light. 
Direct  sunlight  may  be  used.  The  condenser  must  be 
accurately  centered.  The  space  between  it  and  the 
slide  is  usually  filled  in  with  immersion  oil  but  water 
answers  almost  as  well  and  makes  cleaning  easier.  For 
this  work  the  aperture  of  the  oil-immersion  objective 
must  be  reduced  by  placing  in  it  a  "funnel  stop" 
obtainable  from  the  maker  of  the  objective. 

The  chief  use  of  dark-ground  illumination  in  clinical 
work  is  for  demonstration  of  Treponema  pallidum  in 
fresh  material  (see  Fig.  221). 

In  the  "  ultramicroscope "  dark-ground  illumination 
by  means  of  ultra-violet  light  is  utilized.  The  image 
is  invisible  to  the  eye  and  must  be  obtained  by 
photography. 

The  Condenser. — For  the  work  of  the  clinical  labora- 
tory a  substage  condenser  is  a  necessity.  Its  purpose 
is  to  condense  the  light  upon  the  object  to  be  examined. 
For  critical  work  the  light  must  be  focused  on  the  object 
by  raising  or  lowering  the  condenser  by  means  of  the 
screw  provided  for  the  purpose.  The  image  of  the  light 
source  will  then  appear  in  the  plane  of  the  object.  This 
is  best  seen  by  using  a  low-power  objective  and  ocular. 


26  INTRODUCTION 

Should  the  image  of  the  window-frame  or  other  nearby 
object  appear  in  the  field  and  prove  annoying,  the  con- 
denser may  be  raised  or  lowered  a  little.  It  is  often 
advised  to  remove  the  condenser  for  certain  kinds  of 
work,  but  this  is  not  necessary  and  is  seldom  desirable 
in  the  clinical  laboratory. 

The  condenser  is  constructed  for  parallel  rays  of  light. 
With  daylight,  therefore,  the  plane  mirror  should  be 
used;  while  for  the  divergent  rays  of  ordinary  artificial 
light  the  concave  mirror,  which  tends  to  bring  the  rays 
together,  is  best. 

It  is  very  important  that  the  condenser  be  accurately 
centered  in  the  optical  axis  of  the  instrument,  and  most 
high-grade  instruments  have  centering  screws  by  which 
it  can  be  adjusted  at  any  time.  The  simplest  way  to 
recognize  whether  the  condenser  is  centered  is  to  close 
the  diaphragm  beneath  it  to  as  small  an  opening  as 
possible,  then  remove  the  eye-piece  and  look  down  the 
tube.  If  the  diaphragm  opening  does  not  appear  in  the 
center  of  the  field,  the  condenser  is  out  of  center. 

The  use  of  the  condenser  is  further  discussed  in  the 
following  sections. 

Objectives  and  Eye -pieces. — Unfortunately,  different 
makers  use  different  systems  of  designating  their  lenses. 
The  best  system,  and  the  one  chiefly  used  in  this  coun- 
try, is  to  designate  objectives  by  their  focal  lengths  in 
millimeters,  and  eye-pieces  by  their  magnifying  power, 
indicated  by  an  "X."  Most  foreign  makers  use  this 
system  for  their  high-grade  lenses,  but  still  cling  to 
arbitrary  letters  or  numbers  for  their  ordinary  output. 

Objectives  are  of  two  classes — achromatic  and  apo- 
chromatic.     Those  in  general  use  are  of  the  achromatic 


USE    OF   THE    MICROSCOPE  27 

type,  and  they  fulfil  all  requirements  for  ordinary  work. 
Apochromatic  objectives  are  more  highly  corrected  for 
chromatic  and  spheric  aberration,  and  represent  the 
highest  type  of  microscope  lenses  produced.  They  are 
very  desirable  for  photomicrography  and  research, 
but  for  routine  laboratory  work  do  not  offer  advantages 
commensurate  with  their  great  cost.  They  require  the 
use  of  special  "compensating"  eye-pieces. 

Objectives  aje  "corrected"  for  use  under  certain 
fixed  conditions,  and  iliey  will  give  the  best  results  only 
when  used  under  the  conditions  for  which  corrected.  The 
most  important  corrections  are:  {a)  For  tube  length; 
(b)  for  thickness  of  cover-glass;  and  (c)  for  the  medium 
between  objective  and  cover-glass. 

{a)  The  tube  length  with  which  an  objective  is  to  be 
used  is  usually  engraved  upon  it — in  most  cases  it  is 
160  mm.  The  draw-tube  of  the  microscope  should  be 
pulled  out  until  the  proper  length  is  obtained,  as  indi- 
cated by  the  graduations  on  its  side.  When  a  nose- 
piece  is  used,  it  adds  about  15  mm.  to  the  tube  length, 
and  the  draw-tube  must  be  pushed  in  for  that  distance, 
unless,  as  is  the  case  with  the  newer  American  instru- 
ments, the  graduations  upon  the  draw  tube  are  correct 
with  the  nose-piece  in  place. 

{b)  The  average  No.  2  cover-glass  is  about  the  thick- 
ness for  which  most  objectives  are  corrected — usually 
0.17  or  0.18  mm.  One  can  get  about  the  right  thick- 
ness by  buying  No.  2  covers  and  discarding  the  thick 
ones;  or  by  buying  No.  i  covers  and  discarding  the  thin- 
ner ones.  Slight  differences  in  cover-glass  thickness 
can  be  compensated  by  increasing  the  length  of  tube 
wheri  the  cover  is  too  thin,  and  decreasing  it  when  the 


28  INTRODUCTION 

cover  is  too  thick.  This  should  be  done  with  a  spiral 
motion  while  supporting  the  body-tube  with  the  other 
hand.  The  amount  of  correction  necessary  will  de- 
pend upon  the  focal  length  and  numeric  aperture  of  the 
objective.  With  a  4-mm.  objective  of  0.85  numeric 
aperture  a  difference  of  0.03  mm.  in  cover-glass  thick- 
ness requires  a  change  of  30  mm.  in  the  tube  length. 
Many  high-grade  objectives  are  supplied  with  a  "cor- 
rection collar,"  which  accomplishes  the  same  end. 
While  for  critical  work,  especially  with  apochromatics, 
cover-glass  thickness  is  very  important,  one  pays  little 
attention  to  it  in  the  clinical  laboratory.  A  high-power 
dry  lens  always  requires  a  cover,  but  its  exact  thickness 
is  unimportant  in  routine  work.  Very  low-power  and 
oil-immersion  objectives  may  be  used  without  any 
cover-glass. 

(c)  The  correction  for  the  medium  between  objective 
and  cover-glass  is  very  important.  This  medium  may 
be  either  air  or  some  fluid,  and  the  objective  is  hence 
either  a  "dry"  or  an  "immersion"  objective.  The  im- 
mersion fluid  generally  used  is  an  especially  prepared 
cedar  oil,  which  gives  great  optical  advantages  because 
its  index  of  refraction  is  the  same  as  that  of  crown  glass. 
It  is  obvious  that  only  objectives  with  very  short 
working  distance,  as  the  2  mm.,  can  be  used  with  an 
immersion  fluid. 

To  use  an  oil-immersion  objective  a  suitable  field 
for  study  should  first  be  found  with  the  low  power.  A 
drop  of  immersion  oil  is  then  placed  upon  the  cover,  and 
the  objective  lowered  into  it.  A  slight  flash  of  light 
will  be  seen  when  the  front  lens  touches  the  oil.  The 
objective  is  then  brought  to  a  focus  in  the  usual  way. 


USE    OF   THE    MICROSCOPE  29 

In  order  to  avoid  bubbles  the  oil  must  be  placed  upon 
the  cover  carefully  and  without  stirring  it  about. 
Bubbles  are  a  frequent  source  of  trouble,  and  should 
always  be  looked  for  when  an  immersion  objective 
does  poor  work.  They  are  readily  seen  by  removing 
the  eye-piece  and  looking  down  the  tube..  If  they  are 
present,  the  oil  must  be  removed  and  a  new  drop  ap- 
plied. Immediately  after  use  both  objective  and  slide 
should  be  wiped  clean  with  lens-paper  or  a  soft  linen 
handkerchief.  In  an  emergency  glycerin  may  be  used 
instead  of  cedar  oil,  but,  of  course,  with  inferior  results. 

Curvature  of  field,  through  which  it  is  impossible  to 
focus  both  center  and  periphery  sharply  at  the  same 
time,  is  a  very  noticeable  defect;  but  it  is  less  serious 
than  appears  at  first  sight,  particularly  for  visual  work. 
It  is  easily  compensated  by  frequent  use  of  the  fine  fo- 
cusing adjustment.  Complete  flatness  of  field  cannot 
be  attained  without  sacrifice  of  other  and  more  desirable 
properties.  Some  of  the  finest  objectives  made,  notably 
the  apochromatics,  show  decided  curvature. 

The  working  distance  of  an  objective  should  not 
be  confused  with  its  focal  distance.  The  former  term 
refers  to  the  distance  between  the  front  lens  of  the  ob- 
jective, when  it  is  in  focus,  and  the  cover-glass.  It  is 
always  less  than  the  focal  distance,  since  the  "focal 
point"  lies  somewhere  within  the  objective;  and  it 
varies  considerably  with  different  makes.  Long  work- 
ing distance  is  a  very  desirable  feature.  Some  oil-im- 
mersion objectives  have  such  short  working  distance 
that  only  very  thin  cover-glasses  can  be  used. 

A  useful  pointer  can  be  made  by  placing  a  straight 
piece  of  a  hair  across  the  opening  of  the  diaphragm  of 


3©  rNTRODUCTION 

the  eye-piece,  cementing  one  end  with  a  tiny  drop  of 
balsam,  and  cutting  the  hair  in  two  in  the  middle  with 
small  scissors.  WTien  the  eye-piece  is  in  place,  the  hair 
appears  as  a  black  line  extending  from  the  periphery  to 
the  center  of  the  microscopic  field.  If  the  p>ointer  does 
not  appear  sharply  defined  it  is  out  of  focus  and  the 
diaphragm  must  be  raised  or  lowered  a  little  within  the 
ocular. 

The  formation  of  the  microscopic  image  demands 
brief  consideration  (Fig.  7) .  The  rays  of  light  which  are 
reflected  upward  from  the  mirror  and  which  pass 
through  the  object  are  brought  to  a  focus  in  a  magnified, 
inverted  real  image.  This  can  be  focused  to  app>ear  at 
different  levels,  but  when  the  microscope  is  used  in  the 
ordinary*  way  it  is  formed  at  about  the  level  of  the  dia- 
phragm in  the  ocular.  It  can  be  seen  by  remo\-ing  the 
ocular,  placing  a  piece  of  ground  glass  on  the  top  of  the 
tube,  and  focusing  upon  it.  WTien  x^ie-wing  this  image 
a  roll  of  paper  or  a  cylindric  mailing  tube  should  be 
used  to  exclude  extraneous  light.  This  image,  in  turn, 
is  magnified  by  the  eye-lens  of  the  ocular,  producing  a 
second  real  image,  which  is  again  inverted,  and,  there- 
fore, shows  the  object  right  side  up.  This  can  be  seen 
upon  a  groimd  glass  held  a  few  inches  above  the  ocular, 
pro\-ided  strong  artificial  light  be  used  and  the  room 
darkened.  The  eye,  when  it  looks  into  the  microscof>e, 
sees,  not  this  real  image,  but  rather  an  inverted  virtual 
image  which  appears  about  250  mm.  (10  inches)  in  front 
of  the  eye. 

Nmneric  Aperture. — This  expression,  usually  written 
N.A.,  indicates  the  amoimt  of  light  which  enters  an 
objective  from  a  point  in  the  microscopic  field.     In 


USE    OF    THE    MICROSCOPE 


31 


optical  language,  N.A.  is  the  sine  of  one-half  the  angle  of 
aperture  multiplied  by  the  index  of  refraction  of  the 
medium  between  the  cover  and  the  front  lens.     Nu- 


FlG.  7. — Diagram  Showing  Path  of  Light  Rays;  Fi,  Upper  focal 

plane  of  objective;  Fj,  lower  focal  plane  of  eye-piece;  A.  optical  tube 
length  =  distance  between  Fi  and  F2;  Ou  object;  Qj.  real  image  in  F*. 
transposed  by  the  collective  lens,  to  O3.  real  image  in  eye-piece  dia- 
phragm; O4,  virtual  image  formed  at  the  projection  distance  C.  250 
mm.  from  EP.  eyepoint;  CD,  condenser  diaphragm;  L.  mechanical  tube 
length  (160  mm.);  i,  2.  3.  three  pencils  of  parallel  light  coming  from 
different  points  of  a  distant  illuminant,  for  instance,  a  white  cloud, 
which  nintninate  three  diffesent  points  of  the  object. 


32  INTRODUCTION 

meric  aperture  is  extremely  important,  because  upon  it 
depends  resolving  power,  which  is  the  most  important 
property  of  an  objective.^ 

Resolving  power  is  the  ability  to  separate  minute 
details  of  structure.  For  example,  the  dark  portions  of 
a  good  half-tone  picture  appear  gray  or  black  to  the  un- 
aided eye,  but  a  lens  easily  resolves  this  apparently 
uniform  surface  into  a  series  of  separate  dots.  Resolv- 
ing power  does  not  depend  upon  magnification.  The 
fine  lines  and  dots  upon  certain  diatoms  may  be  brought 
out  clearly  and  crisply  (i.e.,  they  are  resolved)  by  an 
objective  of  high  numeric  aperture,  whereas  with  an  ob- 
jective of  lower  numeric  aperture,  but  greater  magnify- 
ing power,  the  same  diatom  may  appear  to  have  a 
smooth  surface,  with  no  markings  at  all,  no  matter  how 
greatly  it  is  magnified.  Knowing  the  N.A.,  it  is  possible 
to  calculate  how  closely  lines  and  dots  may  lie  and  still 
be  resolved  by  a  given  objective.  To  state  the  numeric 
aperture,  therefore,  is  to  tell  what  the  objective  can 
accomplish,  provided,  of  course,  that  spheric  and  chro- 
matic aberrations  are  satisfactorily  corrected.  An  ob- 
jective's N.A.  is  usually  engraved  upon  the  mounting. 

It  is  an  important  fact,  and  one  almost  universally 
overlooked  by  practical  microscopists,  that  the  pro- 
portion of  the  numeric  aperture  of  an  objective  which  is 
utilized  depends  upon  the  aperture  of  the  cone  of  light 
delivered  by  the  condenser.  In  practice,  the  numeric 
aperture  of  an  objective  is  reduced  nearly  to  that  of 

^  Resolving  power  really  depends  upon  two  factors,  the  N.  A.  and  the 
wave  length  of  light,  but  the  latter  can  be  ignored  in  practice.  The 
great  resolving  power  of  the  ultramicroscope  depends  upon  its  use  of 
light  of  short  wave  length. 


USE    OF    THE    IVIICROSCOPE  ^^ 

the  condenser  (which  is  indicated  by  lower-case  letters, 
n.a.).^  The  condenser  should,  therefore,  have  a  nu- 
meric aperture  at  least  equal  to  that  of  the  objective 
with  which  it  is  to  be  used.  Lowering  the  condenser 
below  its  focal  distance  and  closing  the  diaphragm  be- 
neath it  have  the  effect  of  reducing  its  working  aperture. 
A  condenser,  whatever  its  numeric  aperture,  cannot 
deliver  through  the  air  a  cone  of  light  of  greater  N.A. 
than  I.  From  these  considerations  it  follows  that  the 
proper  adjustment  of  the  substage  condenser  is  a  matter 
of  great  importance  when  using  objectives  of  high  N.A., 
and  that,  to  gain  the  full  benefit  of  the  resolving  power 
of  such  objectives,  the  condenser  must  be  focused  on  the 
object  under  examination,  it  must  be  oiled  to  the  under 
surface  of  the  slide  in  the  same  way  as  the  immersion 
objective  is  oiled  to  the  cover-glass,  and  the  substage 
diaphragm  must  be  wide  open.  The  last  condition  in- 
troduces a  difficulty  in  that  colorless  structures  will  ap- 
pear "fogged"  in  a  glare  of  the  light,  making  a  satis- 
factory image  impossible  when  the  diaphragm  is  more 
than  three-quarters  open  (see  Fig.  6).  Wright  suggests 
that  the  size  of  the  light  source  be  so  regulated  by 
a  diaphragm  that  its  image,  thrown  on  the  slide  by 
the  condenser,  coincides  with  the  real  field  of  the  objec- 
tive, and  maintains  that  in  this  way  it  is  possible  to 
reduce  the  glare  of  light  and  to  dispel  the  fog  without 
closing  the  diaphragm. 

One  can  easily  determine  how  much  of  the  aperture 
of  an  objective  is  in  use  by  removing  the  eye-piece,  look- 

^  The  N.A.  of  the  objective  is  not  reduced  wholly  to  that  of  the  con- 
denser, because,  owing  to  diffraction  phenomena,  a  small  part  of  the  un- 
illuminated  portion  of  the  back  lens  is  utilized. 
3 


34  INTRODUCTION 

ing  down  the  tube,  and  observing  what  proportion  of  the 
back  lens  of  the  objective  is  illuminated.  The  relation 
of  the  illuminated  central  portion  to  the  unilluminated 
peripheral  zone  indicates  the  proportion  of  the  numeric 
aperture  in  use.  The  effect  of  raising  and  lowering  the 
condenser  and  of  oiling  it  to  the  slide  can  thus  be  easily 
seen. 

Another  property  of  an  objective  which  depends 
largely  upon  N.A.  is  depth  of  focus,  the  ability  to  render 
details  in  different  planes  clearly  at  the  same  time.  The 
higher  the  N.A.  and  the  greater  the  magnification,  the 
less  the  depth  of  focus.  Any  two  objectives  of  the  same 
focal  length  and  same  N.A.  will  have  exactly  the  same 
depth  of  focus.  Depth  of  focus  can  be  increased  by 
closing  down  the  diaphragm,  and  thus  reducing  the 
N.A.  Great  depth  is  desirable  for  certain  low-power 
work,  but  for  high  powers  it  does  not  offer  advantages 
to  balance  the  loss  of  N.A.  by  which  it  is  attained.  In 
some  cases,  indeed,  it  is  a  real  disadvantage. 

Magnification.— The  degree  of  magnification  should 
always  be  expressed  in  diameters,  not  times,  which  is  a 
misleading  term.  The  former  refers  to  increase  of 
diameter;  the  latter,  to  increase  of  area.  The  compara- 
tively low  magnification  of  loo  diameters  is  the  same  as 
the  apparently  enormous  magnification  of  10,000  times. 

According  to  the  system  of  rating  magnification  in 
use  in  this  country  the  magnifying  power  of  an  objec- 
tive is  ascertained  by  dividing  the  optical  tube-length  by 
the  focal  length  of  the  objective.  The  optical  tube- 
length  is  usually  somewhere  near  165  mm.,  but  it  varies 
with  the  different  objectives  and  the  makers'  catalogs 
must  be  consulted  for  an  accurate  statement  of  magni- 


USE   OF   THE   MICROSCOPE  35 

fying  power.  One  maker,  at  least,  follows  the  commend- 
able plan  of  engraving  both  the  focal  length  of  the 
objective  and  its  initial  magnification  upon  its  barrel. 
This  system  of  rating  magnification  measures  the 
enlarged  image  at  the  level  of  the  diaphragm  in  the 
ocular,  and  this  image  is  in  turn  magnified  by  the  ocular 
so  that  when  an  objective  and  ocular  are  used  together 
the  total  magnification  is  the  product  of  the  two.  In 
the  case,  for  example,  of  the  1.9  mm.  oiL  immersion 
objective,  whose  initial  magnification  is  95  diameters, 
the  total  magnification  with  the  5  X  ocular  is  475  diame- 
ters. These  figures  hold  good,  however,  only  when 
the  ocular  is  rated  upon  the  same  system  as  the  objec- 
tive; thus,  the  4X  ocular  of  the  Zeiss  firm,  which  uses 
a  different  system,  is  equivalent  to  a  6X  ocular  of 
American  make. 

It  is  easy  to  find  the  magnifying  power  of  any  combina- 
tion of  objective  and  ocular  by  actual  trial.  Place  the 
counting  slide  of  the  hemacytometer  upon  the  microscope 
and  focus  the  ruled  lines.  Now  adjust  a  sheet  of  paper  upon 
the  table  close  to  the  microscope  in  such  a  position  that 
when  the  left  eye  is  in  its  proper  place  at  the  ocular  the  paper 
will  lie  in  front  of  the  right  eye  at  the  normal  visual  distance, 
i.e.,  250  mm.  (10  inches).  (The  paper  may  be  supported 
upon  a  book,  if  necessary.)  If  both  eyes  are  kept  open,  the 
ruled  fines  will  appear  to  be  projected  on  the  paper.  With 
a  pencil,  mark  on  the  paper  the  apparent  location  of  the 
lines  which  bound  the  small  squares  used  in  counting  red 
blood  corpuscles  and  measure  the  distance  between  the 
marks.  Divide  this  distance  by  0.05  mm.,  which  is  the 
actual  distance  between  the  lines  on  the  slide.  The 
quotient  gives  the  magnification.  If,  to  take  an  example, 
the  lines  in  the  image  on  the  paper  are  5  mm.  apart,  the 


36  INTRODUCTION 

magnification  is  lOO  diameters.  The  figures  obtained  in 
this  way  will  vary  somewhat  as  one  is  near  or  far  sighted, 
unless  the  defect  of  vision  is  corrected  with  glasses. 

In  practice,  magnification  can  be  increased  in  one  of 
three  ways: 

(a)  Drawing  Out  the  Tube. — Since  the  increased  tube 
length  interferes  with  spheric  correction,  it  should  be 
used  only  with  the  knowledge  that  an  imperfect  image 
will  result. 

(b)  Using  a  Higher  Power  Objective. — As  a  rule,  this 
is  the  best  way,  because  resolving  power  is  also  in- 
creased; but  it  is  often  undesirable  because  of  the  shorter 
working  distance,  and  because  the  higher  objective  often 
gives  greater  magnification  than  is  desired,  or  cuts  down 
the  size  of  the  real  field  to  too  great  an  extent. 

(c)  Using  a  Shorter  Eye-piece. — This  is  the  simplest 
method.  It  has,  however,  certain  limitations.  When 
too  high  an  eye-piece  is  used,  there  results  a  hazy  image 
in  which  no  structural  detail  is  seen  clearly.  This  is 
called  "empty  magnification,"  and  depends  upon  the 
fact  that  the  objective  has  not  sufficient  resolving  power 
to  support  the  high  magnification.  It  has  been  aptly 
compared  to  the  enlargement,  by  stretching  in  all  direc- 
tions, of  a  picture  drawn  upon  a  sheet  of  rubber.  No 
new  detail  is  added,  no  matter  how  great  the  enlarge- 
ment. The  extent  to  which  magnification  can  be  satis- 
factorily increased  by  eye-piecing  depends  wholly  upon 
the  resolving  power  of  the  objective,  and  consequently 
upon  the  N.A.  The  greatest  total  or  combined  magni- 
fication which  will  give  an  absolutely  crisp  picture  is 
found  by  multiplying  the  N.A.  of  an  objective  by  400. 
The  greatest  magnification  which  can  be  used  at  all 


USE    OF   THE    MICROSCOPE  37 

satisfactorily  is  looo  times  the  N.A.  For  example: 
The  ordinary  1.9-mm.  objective  has  a  N.A.  of  1.30;  the 
greatest  magnification  which  will  give  an  absolutely 
sharp  picture  is  520  diameters,  which  is  obtained  ap- 
proximately by  using  a  5.5 X  eye-piece.  Higher  eye- 
pieces can  be  used,  up  to  a  total  magnification  of  1300 
diameters  (12.5X  eye-piece),  beyond  which  the  image 
becomes  wholly  unsatisfactory. 

The  Microscope  in  Use.— Optically,  it  is  a  matter  of 
indifference  whether  the  instrument  be  used  in  the 
vertical  position  or  inclined.  Examination  of  fluids  re- 
quires the  horizontal  stage,  and  since  much  of  the  work 
of  the  clinical  laboratory  is  of  this  nature  it  is  well  to 
accustom  one's  self  to  the  use  of  the  vertical  microscope. 
While  working  one  should  sit  as  nearly  upright  as  is 
possible  compatible  with  comfort,  and  the  height  of 
the  seat  should  be  adjusted  with  this  in  view. 

It  is  always  best  to  "focus  up,"  which  saves  annoy- 
ance and  probable  damage  to  slides  and  objectives. 
This  is  accomplished  by  bringing  the  objective  nearer 
the  slide  than  the  proper  focus,  and  then,  with  the  eye 
at  the  eye-piece,  turning  the  tube  up  until  the  object 
is  clearly  seen.  The  fine  adjustment  should  he  used  only 
to  get  an  exact  focus  with  the  higher  power  objectives  after 
the  instrument  is  in  approximate  focus.  It  should  not  be 
turned  more  than  one  revolution. 

There  will  be  less  fatigue  to  the  eyes  if  both  are  kept 
open  while  using  the  microscope,  and  if  no  effort  is  made 
to  see  objects  which  are  out  of  distinct  focus.  Fine 
focusing  should  be  done  with  the  fine  adjustment,  not 
with  the  eye.     An  experienced  microscopist  keeps  his 


^S  INTRODUCTION 

fingers  almost  constantly  upon  one  or  other  of  the  focus- 
ing adjustments. 

Although  the  ability  to  use  the  eyes  interchangeably 
is  sometimes  very  desirable,  greater  skill  in  recognizing 
objects  will  be  acquired  if  the  same  eye  be  always  used. 
The  left  eye  is  the  more  convenient,  because  the  right 
eye  is  thus  left  free  to  observe  the  drawing  one  may  wish 
to  do  with  the  right  hand.  After  a  little  practice  one 
can  cause  the  microscopic  image  to  appear  as  if  pro- 
jected upon  a  sheet  of  paper  placed  close  to  the  micro- 
scope under  the  free  eye.  This  gives  the  effect  of  a 
camera  lucida,  and  it  becomes  very  easy  to  trace  out- 
lines. When  one  is  accustomed  to  spectacles,  they 
should  not  be  removed. 

It  is  very  desirable  that  one  train  himself  to  work 
with  the  low-power  objective  as  much  as  possible, 
reserving  the  higher  powers  for  detailed  study  of  the 
objects  which  the  low  power  has  found.  This  makes 
both  for  speed  and  for  accuracy.  A  search  for  tube- 
casts,  for  example,  with  the  4-mm.  objective  is  both 
time-consuming  and  liable  to  failure.  Even  such 
minute  structures  as  nucleated  red  corpuscles  in  a 
•stained  blood-film  are  more  quickly  found  with  an 
8-mm.  or  even  a  i6-mm.  objective  combined  with  a 
high  ocular  than  with  the  oil-immersion  lens. 

To  be  seen  most  clearly,  an  object  should  be  brought 
to  the  center  of  the  field.  Acuity  of  vision  will  be 
greatly  enhanced  and  fatigue  lessened  if  all  light  except 
that  which  enters  through  the  microscope  be  excluded 
from  both  eyes.  To  this  end  various  eye-shades  have 
been  devised  and  some  workers  go  so  far  as  to  work 
inside  a  small  tent  constructed  of  strips  of  wood  covered 


USE    OF    THE    MICROSCOPE  39 

with  black  cloth,  the  source  of  illumination  being  placed 
outside  the  tent. 

One  often  wishes  to  mark  a  particular  field  upon  a 
permanent  preparation  so  as  to  refer  to  it  again.  The 
vernier  of  the  mechanical  stage  cannot  be  relied  upon, 
because  it  is  impossible  to  replace  the  stage  in  exactly 
the  same  position  after  it  has  been  removed  and  because 
its  position  is  frequently  changed  by  the  slight  knocks 
which  it  receives.  There  are  on  the  market  several 
"object  markers "  by  which  a  desired  field  can  be  marked 
with  ink,  or  by  a  circle  scratched  on  the  cover-glass 
by  a  minute  diamond,  while  the  slide  is  in  place  on  the 
microscope.  The  circle  is  easily  located  with  a  low 
power.  In  the  absence  of  these,  one  can,  while  using  the 
low  power,  place  minute  spots  with  a  fine  pen  at  the 
edge  of  the  field  on  opposite  sides. 

A  good  marking  material  is  a  cement  which  the  author 
has  long  used  for  making  cells,  ringing  cover-glasses,  etc. 
To  a  few  ounces  of  white  shellac  in  wood  alcohol  add  an  equal 
volume  of  gasoline,  shake  thoroughly,  and  let  stand  for 
twenty-four  hours,  or  until  well  separated  into  two  layers. 
Pipet  off  the  clear  lower  portion,  add  5  to  lo  drops  of  castor 
oil  to  each  ounce,  and  color  with  any  analin  dye  dissolved 
in  absolute  alcohol.  When  too  thick,  thin  with  alcohol. 
This  makes  a  beautiful,  transparent,  easy-flowing  cement 
which  does  not  crack  and  which  is  not  readily  attacked 
by  xylol.  Glycerin  mounts  which  the  writer  ringed  with 
it  twenty  years  ago  are  still  in  perfect  condition. 

Many  good  workers  advise  against  the  use  of  spring 
clips  to  hold  the  slide  against  the  stage  of  the  microscope. 
Manipulation  of  the  slide  with  the  fingers  alone  certainly 
gives  good  training  in  deUcacy  of  touch,  and  is  desirable 


40  INTRODUCTION 

when  examining  infectious  material  which  might  con- 
taminate the  cHps,  or  when  one  must  detect  slight 
pressure  of  the  objective  upon  the  cover-glass  as  in 
studying  a  hanging-drop  preparation.  For  the  ma- 
jority of  examinations,  however,  it  is  more  satisfactory 
to  use  a  clip  at  one  end  of  the  slide,  with  just  sufficient 
pressure  to  hold  the  slide  without  interfering  with  its 
freedom  of  movement. 

Occasionally  when  one  wishes  a  very  low-power  ob- 
jective for  some  special  work  it  may  be  desirable  to 
unscrew  the  front  lens  of  the  i6-mm.  objective  and 
use  the  back  lens  only.  This  procedure  is  not  recom- 
mended for  critical  work,  and  it  should  not  be  tried 
with  high-power  objectives,  which  must  never  he  taken 
apart. 

To  attach  an  objective  it  should  be  supported  in 
position  against  the  nose-piece  by  means  of  the  index- 
finger  and  middle  finger,  which  grasp  it  as  one  would  a 
cigar.  It  is  then  screwed  into  place  with  the  fingers  of 
the  other  hand. 

>  Care  of  the  Microscope. — The  microscope  is  a  deh- 
cate  instrument  and  should  be  handled  accordingly. 
Even  slight  disturbance  of  its  adjustments  may  cause 
serious  trouble.  It  is  so  heavy  that  one  is  apt  to  forget 
that  parts  of  it  are  fragile.  It  seems  unnecessary  to  say 
that  when  there  is  unusual  resistance  to  any  manipula- 
tion, force  should  never  be  used  to  overcome  it  until  its 
cause  has  first  been  sought;  and  yet  it  is  no  uncommon 
thing  to  see  students,  and  even  graduates,  push  a  high- 
power  objective  against  a  microscopic  preparation  with 
such  force  as  to  break  not  only  the  cover-glass,  but  even 
a  heavy  slide. 


USE   OF   THE    MICROSCOPE  4 1 

It  Is  most  convenient  to  carry  a  microscope  with  the 
fingers  grasping  the  pillar  and  the  arm  which  holds  the 
tube;  but  since  this  throws  a  strain  upon  the  fine  adjust- 
ment, it  is  safer  to  carry  it  by  the  base.  In  the  more 
recent  instruments  a  convenient  handle-arm  is  provided. 
To  bend  the  instrument  at  the  joint,  the  force  should  be 
applied  to  the  pillar  and  never  to  the  tube  or  the  stage. 

The  microscope  should  be  kept  scrupulously  clean, 
and  dust  must  not  be  allowed  to  settle  upon  it.  When 
not  in  use  the  instrument  should  be  kept  in  its  case  or 
under  a  cover.  An  expensive  glass  bell-jar  is  not 
needed,  and,  in  fact,  is  undesirable,  except  for  display. 
It  is  heavy  and  awkward  to  handle,  and  when  lifted  is 
almost  certain  (unless  great  care  is  exercised)  to  strike 
the  microscope.  It  is  particularly  liable  to  strike  the 
mechanical  stage  and  disturb  its  adjustment.  The 
simplest,  cheapest,  lightest,  and  probably  the  best  cover 
for  the  microscope  is  a  truncated  cone  or  pyramid  of 
pasteboard,  covered  with  creton  or  similar  material. 
This  is  easily  made  at  home.  In  the  absence  of  a 
special  cover  a  square  of  lintless  cloth  may  be  draped 
over  the  microscope. 

Lens  surfaces  which  have  been  exposed  to  dust  only 
should  be  cleaned  with  a  camel's-hair  brush.  A  small 
brush  and  a  booklet  of  lens-paper  should  always  be  at 
hand  in  the  microscope  case.  Those  surfaces  which  are 
exposed  to  finger-marks  should  be  cleaned  with  lens- 
paper,  or  a  soft  linen  handkerchief,  moistened  with  water 
if  necessary.  The  rubbing  should  be  done  very  gently 
and  with  a  circular  motion.  Particles  of  dirt  which 
are  seen  in  the  field  are  upon  the  slide,  the  eye-piece, 
or  the  condenser.     Their  location  can  be  determined 


42  INTRODUCTION 

by  moving  the  slide,  rotating  the  eye-piece,  and  lower- 
ing the  condenser.  Dirt  upon  the  objective  cannot  be 
seen  as  such;  it  causes  a  diffuse  cloudiness.  When 
the  image  is  hazy,  the  objective  probably  needs  clean- 
ing; or  in  case  of  an  oil-immersion  lens,  there  may  be 
bubbles  in  the  oil. 

Oil  and  balsam  which  have  dried  upon  the  lenses 
— an  insult  from  which  even  dry  objectives  are  not 
immune — may  be  removed  with  alcohol  or  xylol;  but 
these  solvents  must  be  used  sparingly  and  carefully, 
as  there  is  danger  of  softening  the  cement  between  the 
components  of  the  lens.  Some  manufacturers  now 
claim  to  use  a  cement  which  resists  xylol.  Care  must 
be  taken  not  to  get  any  alcohol  upon  the  brass  parts, 
as  it  will  remove  the  lacquer.  Balsam  and  dried  oil 
are  best  removed  from  the  brass  parts  with  xylol. 

When  the  vulcanite  stage  becomes  brown  and  dis- 
colored the  black  color  can  be  restored  by  rubbing  well 
with  petrolatum. 

Measurement  of  Microscopic  Objects. — Of  the  several 
methods,  the  most  convenient  and  accurate  is  the  use  of 
a  micrometer  eye-piece.  In  its  simplest  form  this  is 
similar  to  an  ordinary  eye-piece,  but  it  has  within  it  a  glass 
disk  upon  which  is  ruled  a  graduated  scale.  When  this 
eye-piece  is  placed  in  the  tube  of  the  microscope,  the 
ruled  lines  appear  in  the  microscopic  field,  and  the  size 
of  an  object  is  readily  determined  in  terms  of  the  divisions 
of  this  scale.  The  value  of  these  divisions  in  millimeters 
manifestly  varies  with  different  magnifications.  Their 
value  must,  therefore,  be  determined  separately  for  each 
objective.  This  is  accomplished  through  use  of  a  stage 
micrometer — a  glass  slide  with  carefully  rujed  scale 


USE   OF   THE   MICROSCOPE 


43 


0-1 


divided  into  subdivisions,  usually  hundredths  of  a  milli- 
meter. The  stage  micrometer  is  placed  upon  the  stage 
of  the  microscope  and  brought  into  focus.  The  tube 
of  the  microscope  is  then  pushed  in  or  pulled  out  until 
two  lines  of  the  one  scale  exactly  coin- 
cide with  two  lines  of  the  other.  From 
the  number  of  divisions  of  the  eye-piece 
scale  which  then  correspond  to  each 
division  of  the  stage  micrometer  the 
value  of  the  former  in  micra  or  in  frac- 
tions of  a  millimeter  is  easily  calculated. 
This  value,  of  course,  holds  good  only  for 
the  objective  and  the  tube-length  with  which 
it  was  found.  The  counting  slide  of  the 
hemacytometer  will  answer  in  place  of  a 
stage  micrometer,  the  lines  which  form 
the  sides  of  the  small  squares  used  in 
counting  red  blood  corpuscles  being  0.05 
mm.  apart.  When  using  the  counting 
chamber  with  an  oil-immersion  lens  a 
cover  must  be  used;  otherwise  the  oil 
will  fill  the  ruled  lines  and  cause  them  to 
disappear.  Any  eye-piece  can  be  con- 
verted into  a  micrometer  eye-piece  by 
placing  a  micrometer  disk — a  small  cir-    ^^ig.  8.— Scale 

11  ,  -1  IT  1  ,     ,    of    the  step    mi- 

cular  glass  plate  with  ruled  scale — ruled  crometer  eye- 
side  down  upon  its  diaphragm.  If  the  ^^^'^^' 
lines  upon  this  are  at  all  hazy  the  disk  has  probably 
been  inserted  upside  down  or  else  the  diaphragm  is 
out  of  its  proper  position.  Usually  it  can  be  pushed 
up  or  down  as  required.  The  new  "step"  micrometer 
eye-piece  is  very  satisfactory.     The  step-hke  arrange- 


100 -K 


-103 


44  INTRODUCTION 

ment  of  the  scale  (Fig.  8)  makes  it  easy  to  read  and 
the  divisions  are  such  that  they  read  in  micra  or  easy 
multiples  of  micra  with  little  or  no  change  from  the 
regular  tube-length. 

The  following  method  of  micrometry  is  less  accurate, 
but  is  fairly  satisfactory  for  comparatively  coarse  objects, 
such  as  the  ova  of  parasites.  A  ruled  scale  corresponding 
to  the  magnified  image  of  the  hemacytometer  ruling  is 
drawn  upon  cardboard  in  the  manner  described  for  as- 
certaining magnifications  (see  p.  35)  except  that  the 
card  is  placed  upon  the  table  beside  the  microscope 
and  not  necessarily  at  a  distance  of  ten  inches  from  the 
eye.  This  card  may  then  be  used  as  a  micrometer, 
and  should  be  inscribed  with  the  value  of  its  gradua- 
tions, and  the  objective,  ocular,  and  tube  length  with 
which  it  is  to  be  used.  In  the  example  cited  upon  p.  35 
the  lines  on  the  card  are  5  mm.  apart,  corresponding 
to  an  actual  distance  of  0.05  mm.  To  measure  an 
object,  the  cardboard  is  placed  in  the  position  which 
it  occupied  when  made  (upon  the  table  at  the  right 
of  the  microscope).  The  lines  and  the  objects  on  the 
slide  can  then  be  seen  together,  and  the  space  cov- 
ered by  any  object  indicates  its  size.  The  graduations 
made  as  above  indicated  are  too  coarse  for  most  work, 
and  they  should  be  subdivided.  If  five  subdivisions 
are  made,  each  will  have  a  value  of  10  ju. 

Tuttle  has  suggested  that  in  feces  and  other  examina- 
tions a  little  lycopodium  powder  be  mixed  with  the 
material.  The  granules  are  of  uniform  size — 30  /x  in 
diameter — and  are  easily  recognized  (Fig.  9).  They 
furnish  a  useful  standard  with  which  the  size  of  other 
structures  can  be  compared.     Care  must  be  exercised 


USE    OF    THE    MICROSCOPE  45 

not  to  use  too  much  powder.  The  lycopodium  is  con- 
veniently kept  in  a  gelatin  capsule,  and  a  faint  cloud  can 
be  dusted  over  the  slide  by  gently  scraping  the  edge  of 
the  lid  upon  the  rim  of  the  capsule. 

The  principal  microscopic  objects  which  are  measured 
clinically  are  animal  parasites  and  their  ova  and  abnor- 
mal blood-corpuscles.  The  metric  system  is  used  almost 
exclusively.  For  very  small  objects  o.ooi  mm.  has  been 
adopted  as  the  unit  of  measurement,  under  the  name 


Fig.  9. — Egg  of    1  tenia  saginala.     Lycopodium  granules  used  as  mi- 
crometer (X  250). 

micron.  It  is  represented  by  the  Greek  letter  ix.  For 
larger  objects,  where  exact  measurement  is  not  essential, 
the  diameter  of  a  red  blood-corpuscle  (7  to  8  n)  is  some- 
times taken  as  a  unit. 

Photomicrography. — Although  high-grade  photo- 
micrography requires  expensive  apparatus  and  con- 
siderable skill  in  its  use,  fairly  good  pictures  of  micro- 
scopic structures  can  be  made  by  any  one  with  simple 
instruments. 


46  INTRODUCTION    . 

Any  camera  with  focusing  screen  or  a  Kodak  with 
plate  attachment  may  be  used.  It  is  best,  but  not  neces- 
sary, to  remove  the  photographic  lens.  The  camera  is 
placed  with  the  lens  (or  lens-opening,  if  the  lens  has  been 
removed)  looking  into  the  eye-piece  of  the  microscope, 
which  may  be  in  either  the  vertical  or  the  horizontal 
position.  One  can  easily  rig  up  a  standard  to  which  the 
camera  can  be  attached  in  the  proper  position  by  means 
of  a  tripod  screw.  A  light-tight  connection  can  be  made 
of  a  cylinder  of  paper  or  a  cloth  sleeve  with  draw-strings. 
The  image  will  be  thrown  upon  the  ground-glass  focusing 
screen,  and  is  focused  by  means  of  the  fine  adjustment  of 
the  microscope.  The  degree  of  magnification  is  ascer- 
tained by  placing  the  ruled  slide  of  the  blood-counting 
instrument  upon  the  microscope  and  measuring  the 
image  on  the  screen.  The  desired  magnification  is 
obtained  by  changing  objectives  or  eye-pieces  or  length- 
ening the  camera-draw. 

Focusing  is  comparatively  easy  with  low  powers,  but 
when  using  an  oil-immersion  objective  it  is  a  difficult 
problem  unless  the  source  of  light  be  very  brilliant.  If 
one  always  uses  the  same  length  of  camera  and  micro- 
scope tube,  a  good  plan  is  as  follows :  Ascertain  by  trial 
with  a  strong  light  how  far  the  fine  adjustment  screw 
must  be  turned  from  the  correct  eye  focus  to  bring  the 
image  into  sharp  focus  upon  the  ground-glass  screen. 
At  any  future  time  one  has  only  to  focus  accurately 
with  the  eye,  bring  the  camera  into  position,  and  turn 
the  fine  adjustment  the  required  distance  to  right  or 
left.  When  the  camera-draw  is  lo  inches  little  or  no 
change  in  the  focusing  adjustment  will  be  necessary. 

The  light  should  be  as  intense  as  possible  in  order  to 


USE    OF    THE    MICROSCOPE  47 

shorten  exposure,  but  any  light  that  is  satisfactory  for 
ordinary  microscopic  work  will  answer.  The  light  must 
be  carefully  centered.  It  is  nearly  always  necessary  to 
insert  a  colored  filter  between  the  light  and  the  micro- 
scope. Pieces  of  colored  window-glass  are  useful  for 
this  purpose  but  much  better  filters  can  be  purchased 
at  trifling  cost.  The  writer  has  had  best  results  with 
the  Wratten  "micro"  filters.  These  may  be  purchased 
in  the  form  of  gelatin  sheets  which  can  be  cemented 
between  glass  plates  with  balsam.  The  screen  should 
have  a  color  complementary  to  that  which  it  is  desired 
to  bring  out  strongly  in  the  photograph :  for  blue  struc- 
tures, a  yellow  screen;  for  red  structures,  a  green  screen. 
For  the  average  stained  preparation,  a  picric-acid 
yellow  or  a  yellow  green  will  be  found  satisfactory. 

Very  fair  pictures  can  be  made  on  Kodak  film,  but 
orthochromatic  plates  (of  which  Cramer's  "Iso"  and 
Seed's  "Ortho"  are  examples)  give  much  better  re- 
sults. Panchromatic  plates  Hke  the  Wratten  "M"  are 
still  better  but  are  more  difficult  to  handle  because 
more  sensitive  to  red  light.  In  order  to  avoid  halation 
all  plates  should  if  possible  be  "backed."  The  length 
of  exposure  depends  upon  so  many  factors  that  it  can 
be  determined  only  by  trial.  It  will  probably  vary 
from  a  few  seconds  to  fifteen  minutes.  Plates  are 
developed  in  the  usual  way.  Either  the  tray  or  tank 
method  may  be  used,  but  in  order  to  secure  good  con- 
trast it  is  often  desirable  to  overdevelop  somewhat. 
Metol-hydrochinon  is  an  excellent  developer,  as  it  gives 
good  contrast  with  full  detail. 

The  photograph  from  which  Fig.  lo  was  made  was 
taken  with  a  Kodak  and  plate  attachment  on  an  "Iso" 


48  INTRODUCTION 

plate,  the  source  of  light  being  the  electric  lamp  and 
condensing  lens  illustrated  in  Fig.  3.  It  was  focused  by 
the  method  described  above.  The  screen  was  a  picric- 
acid  stained  photographic  plate.  Exposure,  three  and  a 
half-minutes.  The  picture  loses  considerable  detail  in 
reproduction. 


Fig.   10. — Leukemic   blood    (about  X  650).      Photograph  taken  with  a 
Kodak,  as  described  in  the  text. 


Choice  of  a  Microscope. — It  is  poor  economy  to  buy 
a  cheap  instrument. 

For  the  work  of  a  clinical  laboratory  the  microscope 
should  preferably  be  of  the  handle-arm  type,  and 
should  have  a  large  stage.  It  should  be  provided  with  a 
substage  condenser  (preferably  of  1.40  n.a.),  three  or 
more  objectives  on  a  revolving  nose-piece,  and  two  or 
more  eye-pieces.  After  one  has  learned  to  use  them, 
the  new  mon-objective  binocular  microscopes  are 
extremely  satisfactory,  giving  an  impression  of  stereo- 
scopic vision. 

The  most  generally  useful  objectives  are:  16  mm., 
4  mm.,  and  2  mm.  oil  immersion.  The  4-mm.  objective 
may  be  obtained  with  N.A.  of  0.65  to  0.85.     If  it  is  to 


^w^ 


USE   OF   THE    MICROSCOPE  49 

be  used  for  blood-counting,  the  former  is  preferable, 
since  its  working  distance  is  sufficient  to  take  the  thick 
cover  of  the  blood-counting  instrument.  For  coarse 
objects  a  32-mm.  objective  is  very  desirable.  The  eye- 
pieces most  frequently  used  are  5X  and  loX.  A  very 
low  power  (2X)  and  a  very  high  (15 X)  will  sometimes 
be  found  useful.  The  microm- 
eter eye-piece  is  almost  a 
necessity.  A  mechanical  stage, 
preferably  of  the  attachable 
type,  is  almost  indispensable 
for  blood  and  certain  other 
work.     A  new,  simple  and  com- 

,•      1       .  .  ,  r         Fig.   II. — An  inexpensive 

paratlVely    mexpensive   stage  of     niechanical  stage  with  rack 

this  type  is  shown  in  Fig.   11.  ^nd   pinion    movement      in 

.  one    direction. 

A  nrst-class  monocular  micro- 
scope, of  either  American  or  foreign  make,  equipped 
as  just  described,  will  cost  in  the  neighborhood  of  $70 
to  $80,  exclusive  of  the  mechanical  stage. 

Practical  Exercises.— The  following  is  a  brief  outline 
of  certain  exercises  which  the  author  has  found  useful 
in  teaching  microscopy.  The  student  must  learn  as 
early  as  possible  what  can  be  expected  of  his  microscope 
with  proper  manipulation.  When  he  sits  down  to 
work  his  first  glance  should  tell  him  whether  the  instru- 
ment is  giving  its  best  results.  If  the  microscopic 
picture  falls  short  of  the  best,  he  must  locate  the  diffi- 
culty and  correct  it  before  proceeding. 

1.  Clean  the  microscope  and  study  its  parts,  familiariz- 
ing yourself  with  the  names,  purposes,  and  movements  of 
each  (see  Fig.  i). 

2.  Practice  the  manipulations  necessary  to  locate  particles 


50  INTRODUCTION 

of  dust  or  dirt  which  appear  in  the  microscopic  field  (see 

p.  41). 

3.  Place  the  microscope  before  a  window,  focus  upon 
a  dusty  slide  and  adjust  condenser  and  mirror  so  that 
the  image  of  the  window-frame  or,  better,  of  trees  just 
outside  the  window  appear  in  the  microscopic  field. 
Try  the  effect  of  raising  and  lowering  the  condenser,  and 
of  changing  from  plane  to  concave  mirror,  upon  these 
images.  Note  that  they  cause  an  unevenly  illuminated 
or  mottled  field  when  a  little  out  of  focus. 

4.  Insert  a  "pointer"  in  one  of  the  oculars  [see  p.  29). 

5.  Study  illumination.  Use  a  slide  of  some  colorless 
structures  such  as  cholesterol  crystals  and  two  prepara- 
tions of  blood,  one  a  dried  film  stained  with  eosin  or  any 
blood  stain  (see  p.307)  and  mounted  in  balsam,  the  other 
an  unstained  wet  preparation  made  is  described  for  the 
malarial  parasite  (see  p.  355).  Study  only  the  areas  in 
which  the  corpuscles  are  well  separated. 

(i)  Place  one  of  these  on  the  microscope,  bring  to  a 
focus,  and  practice  the  manipulations  necessary 
to  secure  (see  p.  22) — 
(a)  Central  illumination. 
(6)  Oblique  illumination, 
(c)  Strong  and  subdued  illumination. 
The  field  in  each  case  must  be  evenly  hghted  throughout, 
without  mottling.     Continue  until  you  can  adjust  any  de- 
sired form  of  illumination  quickly  and  surely,  and  can  recog- 
nize each  by  a  glance  into  the  microscope. 

(2)  Using  the  three  slides  mentioned  above,  ascer- 
tain the  best  form  of  illumination  to  study  (see 

P-  23)— 

(a)  Outlines. 

(6)  Color. 

(c)  Surface  contour.    The  unstained  normal  and 

crenated  red  corpuscles  are  excellent  objects 

for  study  of  surface  contour. 


USE   OF   THE   MICROSCOPE  5 1 

(3)  Try  dark-ground  illumination  by  means  of  the  sub- 
stage  disk  (see  p.  24).  Study  the  unstained  blood- 
smear  and  draw  a  few  corpuscles.  Also  examine 
the  cholesterol  crystals,  a  drop  of  diluted  milk 
and  a  bit  of  lens  paper.  Use  the  i6-mm.  ob- 
jectiv'e  for  this. 

6.  With  central  illumination,  locus  upon  a  slide  and  ob- 
serve how  much  of  the  numeric  aperture  is  in  use  (see  p.  33). 
Try  the  effect  upon  numeric  aperture  of — 

(i)  Opening  and  closing  the  diaphragm. 

(2)  Raising  and  lowering  the  condenser. 

(3)  Using  the  oil-immersion  objective — 
(a)  Without  oil. 

{b)  With  oil  between  objective  and  cover-glass. 
(c)   With  oil  between  slide  and  condenser. 

7.  Upon  the  same  species  of  diatom  compare  two  objec- 
tives of  3-mm.  focus  (therefore  of  same  magnifying  power), 
one  of  N.A.  1.4  and  the  other  of  N.A.  0.85.  They  will  be 
adjusted  by  the  instructor.  Note  the  superior  resolving 
power  of  the  lens  of  high  N.A.  (see  p.  ^^2). 

8.  Practice  using  the  oil-immersion  objective  (see  p.  28) 
upon  an  unstained  film  preparation  of  blood  or  a  slide 
strewn  with  diatoms.  These  are  nearly  colorless  and 
hence  difficult  to  see.  If  there  is  difficulty  in  finding  the 
specimen,  move  the  slide  about  while  lowering  the  objective 
to  a  focus.  Moving  objects  will  catch  the  eye  as  the  ob- 
jective approaches  the  correct  focus.  If  a  cover-glass  is 
used,  its  edge  can  be  easily  found,  but  it  must  be  borne  in 
mind  that  when  the  upper  surface  of  the  cover  is  in 
focus,  objects  beneath  it  are  so  far  out  of  focus  as  to  be 
invisible. 

Produce  some  bubbles  in  the  oil  by  stirring  it  about  on 
the  slide;  observe  their  effect  on  the  image  of  the  blood 
cells  or  diatoms,  and  learn  to  detect  their  presence  (see 
p.  29). 


52  INTRODUCTION 

9.  Image  formation  (see  p.  30).  Mount  a  bit  of  paper 
printed  with  very  small  type  using  oil  or  balsam  to  render 
it  transparent.  Focus  upon  this  with  a  low  power  ob- 
jective. Remove  the  ocular  and  lay  a  piece  of  ground 
glass  across  the  top  of  the  tube.  This  forms  a  screen  upon 
which  an  image  can  be  focused  by  means  of  the  coarse 
adjustment.  Note  whether  it  is  right  side  up  or  re- 
versed. Repeat  this  with  the  ocular  in  place,  holding 
the  ground  glass  some  inches  above  the  ocular.  These 
exercises,  especially  the  last,  are  best  done  in  a  darkened 
room  with  strong  artificial  illumination,  but  extraneous 
light  can  usually  be  sufficiently  excluded  by  viewing 
the  image  through  a  pasteboard  mailing  cylinder. 

10.  Find  by  trial  the  magnification  produced  by  your 
i6-mrn.  objective  with  the  4X  or  6X  ocular  (see  p.  35). 
Compare  your  result  with  that  listed  by  the  maker  of  the 
microscope. 

11.  Micrometry. 

(i)  Evaluate  the  scale  of  your  micrometer  eye-piece 
with  a  high-power  objective,  and  measure  accu- 
rately 10  red  blood  corpuscles  and  10  leuko- 
cytes (see  pp.  42,  43). 

(2)  Prepare  a  cardboard  micrometer  and  measure  10 
lycopodium  granules  (see  p.  44). 

12.  Focus  upon  a  stage  micrometer  or  hemacytometer 
slide  and  measure  the  diameter  of  the  real  field  of  each 
of  your  objectives  with  each  of  the  oculars.  Note  the 
effect  of  increasing  the  tube-length. 

13.  Study  the  following  structures,  chiefly  with  a  view  to 
best  illumination.  Examine  separately  the  color,  outline 
and  surface  contour  of  each.  Many  of  these  are  met  as 
accidental  contaminations  in  microscopic  preparations  and 
one  must  learn  to  recognize  them.    Make  drawings  of  each. 

Fluids  are  examined  by  placing  a  drop  in  the  center  of  a 
clean  slide  and  applying  a  cover-glass.     The  drop  should  be 


USE    OF    THE    MICROSCOPE  53 

large  enough  to  fill  the  space  between  the  slide  and  cover, 
but  not  large  enough  to  float  the  cover  about.  Fibers  or 
insoluble  powder  may  be  placed  in  a  drop  of  water  and 
covered. 

(i)  With  1 6  mm.  and  4  mm.  objectives  examine  the 
upper  surface  of  a  new  cover-glass  without  clean- 
ing. Usually  it  will  show  dirt  and  often  crystals; 
if  not,  make  finger  prints  upon  it  and  produce 
faint  scratches  by  rubbing  two  covers  together. 

(2)  Air  bubbles  produced  by  shaking  a  little  diluted 
mucilage. 

(3)  Fresh  milk  diluted  with  three  or  four  volumes  of 
water.     Prepare  three  slides. 

(a)  Examine  one  untreated. 

(b)  Treat  one  with  solution  of  Sudan  III.  (For 
method  see  p.  201.)  Note  color  assumed  by 
the  fat  globules.  This  is  one  of  the  most 
useful  tests  for  microscopic  fat. 

(c)  Treat  one  with  dilute  acetic  acid.  Note 
clumping  of  globules  similar  to  that  of  ty- 
phoid bacilli  in  the  Widal  test. 

(4)  A  drop  of  diluted  India-ink.  Note  the  dancing 
motion  of  the  smaller  particles  ("Brownian 
motion"). 

(5)  Starch  granules.  Gently  scrape  the  freshly  cut 
surface  of  a  potato  with  a  knife,  place  a  drop 
of  the  cloudy  fluid  upon  a  slide  with  a  drop  of 
water,  and  apply  a  cover-glass.  Make  two 
preparations. 

(a)  Examine  one  untreated.  Note  the  variously 
sized  starch  granules,  oval,  colorless,  concen- 
trically striated.  Make  sure  that  you  find 
the  best  form  of  illumination  to  bring  out 
the  striations  clearly.  The  starch  granules 
themselves  are  easy  to  see  because  of  their 


54  INTEODUCTION 

broad  dark  outlines.  This  means  that  they 
are  "highly  refractive" — a  term  much  used  in 
describing  microscopic  structures — or,  more 
correctly,  that  their  index  of  refraction  dif- 
fers greatly  from  that  of  the  medium  in 
which  they  are  mounted. 

(b)  Treat  one  with  dilute  Lugol's  or  Gram's  iodin 
solution. 

Note  the  change  in  color  of  the  granules. 
This  is  the  standard  test  for  starch. 

(6)  Yeast  which  has  been  growing  in  a  dextrose  solu- 
tion.    Make  two  preparations. 

(a)  Examine  one  unstained.     Note  "budding." 

{b)  Treat   one   with   iodin   solution.     Compare 

color  of  yeast  with  that  taken  by  starch. 

(7)  Mold  from  moldy  food.  Note  hyphae  and  spores. 
Try  the  effect  of  iodin. 

(8)  Various  fibers  and  other  structures  mounted  in  a 

drop  of  water, 
(a)  Cotton. 
lb)  Wool. 

(c)  Linen. 

(d)  Silk. 

(e)  Feather  tip. 

(/)  Some  dust  from  a  carpeted  room.  Colored 
fibers  from  the  carpet  are  frequently  found 
in  urine. 

(g)  A  hair. 

(9)  A  drop  of  decomposing  urine.  Note  bacteria  of 
various  kinds,  some  motile,  some  non-motile. 
Make  an  effort  to  distinguish  true  motility  from 
that  due  to  currents  in  the  fluid  and  to  "Brownian 
motion." 

(10)  Some  of  the  scum  from  the  bottom  of  a  stagnant 
pool.     Note  the  abundance  of  microscopic  life. 


USE    OF    THE   MICROSCOPE  55 

Look  especially  for  diatoms,  amebae  and  ciliated 
organisms. 
(11)  Test  your  proficiency  in  using  the  microscope  by 
trying  to  resolve  diatoms.  For  the  4-mm.  objec- 
tive use  Pleurosigma  angulatum.  The  dots  should 
be  clearly  seen.  For  the  oil-immersion  lens  use 
Surirella  gemma.  The  fine  lines  between  the 
ribs  should  be  seen  as  rows  of  dots.  As  a  most 
critical  test,  both  of  the  oil-immersion  lens  and 
of  your  skill  in  manipulation  use  Amphipleura 
pellucida.  Select  a  diatom  of  large  size.  Use 
oblique  illumination  and  endeavor  to  bring  out 
the  cross  striations.  Try  the  same  with  central 
light,  although  you  are  not  likely  to  succeed. 
These  striations  consist  of  rows  of  extremely 
minute  dots  which  can  be  seen  only  under  the 
most  favorable  conditions  such  as  are  not  at- 
tained in  clinical  work. 


CHAPTER  I 
THE  SPUTUM 

Preliminary  Considerations. — Before  beginning  the 
study  of  the  sputum,  the  student  will  do  well  to  familiar- 
ize himself  with  the  structures  which  may  be  present 
in  the  normal  mouth,  and  which  frequently  appear  in 
the  sputum  as  contaminations.  Nasal  mucus  and  ma- 
terial obtained  by  scraping  the  tongue  and  about  the 
teeth  should  be  studied  as  described  for  unstained  spu- 
tum. A  drop  of  Lugol's  solution  should  then  be  placed 
at  the  edge  of  the  cover,  and,  as  it  runs  under,  the  efifect 
upon  difTerent  structures  noted.  Another  portion 
should  be  spread  upon  slides  or  covers  and  stained  by 
some  simple  stain  and  by  Gram's  method.  The  struc- 
tures likely  to  be  encountered  are  epithelial  cells  of 
columnar  and  squamous  types;  leukocytes,  chiefly 
mononuclear,  the  so-called  salivary  corpuscles;  food- 
particles;  Leptothrix  buccalis;  great  numbers  of  sapro- 
phytic bacteria;  and  frequently  spirochetes  and  enda- 
mebae.     These  structures  are  described  later. 

When  collecting  the  sample  for  examination,  the 
morning  sputum,  or  the  whole  amount  for  twenty-four 
hours  should  be  saved.  In  beginning  tuberculosis 
tubercle  bacilli  can  often  be  found  in  that  first  coughed 
up  in  the  morning  when  they  cannot  be  detected  at  any 
other  time  of  day.  Sometimes,  in  these  early  cases, 
there  are  only  a  few  mucopurulent  flakes  which  contain 

56 


THE    SPUTUM  57 

the  bacilli,  or  only  a  small  purulent  mass  every  few  days, 
and  these  may  easily  be  overlooked  by  the  patient. 

Patients  should  be  instructed  to  rinse  the  mouth 
well  in  order  to  avoid  contamination  with  food-particles 
which  may  prove  confusing  in  the  examination,  and  to 
make  sure  that  the  sputum  comes  from  the  lungs  or 
bronchi  and  not  from  the  nose  and  nasopharynx. 
Many  persons  find  it  difficult  to  distinguish  between 
the  two.  It  is  desirable  that  the  material  be  raised 
with  a  distinct  expulsive  cough,  but  this  is  not  always 
possible.  Material  from  the  upper  air-passages  can 
usually  be  identified  by  the  large  proportion  of  mucus 
and  the  character  of  the  epithelial  cells. 

The  sputum  of  infants  and  young  children  is  usually 
swallowed  and  therefore  cannot  be  collected.  In  such 
cases  examination  of  the  feces  for  tubercle  bacilli  will 
sometimes  establish  a  diagnosis  of  tuberculosis. 

As  a  receptacle  for  the  sputum,  a  clean,  wide-mouthed 
bottle  with  tightly  fitting  cork  may  be  used.  The  pa- 
tient must  be  particularly  cautioned  against  smearing 
any  of  it  upon  the  outside  of  the  bottle.  This  is  prob- 
ably the  chief  source  of  danger  to  those  who  examine 
sputum.  Disinfectants  should  not  be  added.  Al- 
though some  of  them  (phenol,  for  example)  do  not 
interfere  with  detection  of  tubercle  bacilli,  they  gener- 
ally so  alter  the.  character  of  the  sputum  as  to  render 
it  unfit  for  other  examinations. 

The  following  outline  is  suggested  for  the  routine 
examination : 

I.  Spread  the  material  in  a  thin  layer  in  a  large  Petri 
dish  or  between  two  plates  of  glass.  The  use  of  glass 
plates  is  messy,  but  is  to  be  recommended  for  careful  work. 


58  THE   SPUTUM 

The  top  plate  should  be  much  smaller  than  the  lower  one, 
or  have  some  sort  of  handle. 

2.  Examine  all  parts  carefully  with  the  naked  eye  (best 
over  a  black  background)  or  %vith  a  hand  lens.  The  por- 
tions most  suitable  for  further  examination  may  thus  be 
easily  selected.  This  macroscopic  examination  should  never 
be  omitted. 

3.  Transfer  various  portions,  including  all  suspicious 
particles,  to  clean  slides,  cover,  and  examine  unstained  with 
the  microscope  (see  p.  63). 

4.  Slip  the  covers  from  some  or  all  of  the  above  unstained 
preparations,  leaving  a  thin  smear  on  both  slide  and  cover. 

5.  Dr>'  and  fix  the  smears  and  stain  one  or  more  by  each 
of  the  following  methods: 

(a)  For  tubercle  bacilli  (see  p.  76). 
(6)   Gram's  method  (see  p.  572). 

6.  When  indicated,  make  special  examinations  for — 
(a)   Capsules  of  bacteria  (see  p.  87). 

{h)  Eosinophilic  cells  (see  p.  91). 

(c)  Much's  granules  (see  p.  83). 

(d)  Presence  of  albumin  (see  p.  94). 

After  the  examination  the  sputum  must  be  destroyed 
by  heat  or  chemicals,  and  everything  w^hich  has  come  in 
contact  with  it  must  be  sterilized.  The  utmost  care 
must  be  taken  not  to  allow  any  of  it  to  dry  and  become 
disseminated  through  the  air.  If  flies  are  about,  it  must 
be  kept  covered .  It  is  a  good  plan  to  conduct  the  exami- 
nation upon  a  large  newspaper,  which  can  then  be 
burned.  Contamination  of  the  work  table  is  thus 
avoided.  If  this  is  not  feasible,  the  table  should  be 
washed  off  with  10  per  cent,  lysol  or  other  disinfectant 
solution,  and  allowed  to  dry  slowly,  as  soon  as  the 
sputum  work  is  finished. 


PHYSICAL  EXAMINATION  59 

Examination  of  the  sputum  is  most  conveniently  con- 
sidered under  four  heads:  I.  Physical  examination. 
II.  Microscopic  examination.  III.  Chemic  examination. 
IV.  Characteristics  of  the  sputum  in  various  diseases. 

I.    PHYSICAL  EXAMINATION 

1 .  Quantity. — The  quantity  expectorated  in  twenty- 
four  hours  varies  greatly.  It  may  be  so  slight  as  to  be 
overlooked  entirely  in  beginning  tuberculosis.  It  is 
usually  small  in  acute  bronchitis  and  lobar  pneumonia. 
It  may  be  very  large — sometimes  as  much  as  looo  c.c. — 
in  advanced  tuberculosis  with  large  cavities,  edema  of 
the  lung,  bronchiectasis,  and  following  rupture  of  an 
abscess  or  empyema.  It  is  desirable  to  obtain  a 
general  idea  of  the  quantity,  but  accurate  measurement 
is  unnecessary. 

2.  Color. — Since  the  sputum  ordinarily  consists  of 
varying  proportions  of  mucus  and  pus,  it  may  vary 
from  a  colorless,  translucent  mucus  to  an  opaque, 
whitish  or  yellow,  purulent  mass.  A  yellowish  green 
is  frequently  seen  in  advanced  phthisis  and  chronic 
bronchitis.  In  jaundice,  in  caseous  pneumonia,  and  in 
slowly  resolving  lobar  pneumonia  it  may  assume  a 
bright  green  color,  due  to  bile  or  altered  blood-pigment. 

A  red  or  reddish-brown  color  usually  indicates  the 
presence  of  blood.  Bright  red  blood,  most  commonly 
in  streaks,  is  strongly  suggestive  of  phthisis.  It  may 
be  noted  early  in  the  disease  and  generally  denotes  an 
extension  of  the  tuberculous  process.  One  must,  how- 
ever, be  on  his  guard  against  blood-streaked  mucus 
or  muco-pus  originating  in  nasopharyngeal  catarrh. 
Tuberculous   patients   not   infrequently   mistake   this 


6o  THE    SPUTUM 

for  true  sputum.  Blood-stained  sputum  is  also  some- 
times seen  in  bronchiectasis.  A  rusty  red  sputum  is  the 
rule  in  croupous  pneumonia,  and  was  at  one  time  con- 
sidered pathognomonic  of  the  disease.  Exactly  similar 
material  may  be  raised  in  pulmonary  infarction. 
"Prune- juice"  sputum  is  said  to  be  characteristic  of 
"drunkard's  pneumonia."  It  at  least  indicates  a 
dangerous  type  of  the  disease,  as  it  is  apparently 
referable  to  coincident  edema  of  the  lung.  A  brown 
color,  due  to  altered  blood-pigment,  follows  hemor- 
rhages from  the  lungs,  and  is  present,  to  greater  or  less 
degree,  in  chronic  passive  congestion  of  the  lungs, 
which  is  most  frequently  due  to  a  heart  lesion. 

Gray  or  black  sputum  is  observed  among  those  who 
work  much  in  coal-dust,  and  is  occasionally  seen  in 
smokers  who  are  accustomed  to  "inhale." 

3.  Consistence. — According  to  their  consistence, 
sputa  are  usually  classified  as  serous,  mucoid,  purulent, 
seropurulent,  mucopurulent,  etc.,  which  names  explain 
themselves.  As  a  rule,  the  more  mucus  and  the  less  pus 
and  serum  a  sputum  contains,  the  more  tenacious  it  is. 

The  rusty  sputum  of  croupous  pneumonia  is  ex- 
tremely tenacious,  so  that  the  vessel  in  which  it  is  con- 
tained may  be  inverted  without  spilling  it.  The  same 
is  true  of  the  almost  purely  mucoid  sputum  ("sputum 
crudum")  of  beginning  acute  bronchitis,  and  of  that 
which  follows  an  attack  of  asthma.  A  purely  serous 
sputum,  usually  slightly  blood  tinged,  is  fairly  char- 
acteristic of  edema  of  the  lungs. 

Formerly  much  attention  was  paid  to  the  so-called 
''nummular  sputum."  This  consists  of  definite  muco- 
purulent masses  which  flatten  out  into  coin-like  disks 


PHYSICAL    EXAMINATION  6 1 

and  sink  in  water.     It  is  fairly  characteristic  of  ad- 
vanced tuberculosis. 

4.  Dittrich's  Plugs. — While  these  bodies  sometimes 
appear  in  the  sputum,  they  are  more  frequently  expec- 
torated alone.  They  are  caseous  masses,  usually  about 
the  size  of  a  pin-head,  but  sometimes  reaching  that  of 
a  bean.  The  smaller  ones  are  yellow,  the  larger  ones 
gray.  When  crushed,  they  emit  a  foul  odor.  Micro- 
scopically, they  consist  of  granular  debris,  fat-globules, 
fatty  acid  crystals,  and  bacteria.  They  are  formed 
in  the  bronchi,  and  are  sometimes  expectorated  by 
healthy  persons,  but  are  more  frequent  in  putrid  bron- 
chitis and  bronchiectasis.  The  laity  commonly  regard 
them  as  evidence  of  tuberculosis.  The  similar  caseous 
masses  which  are  formed  in  the  crypts  of  the  tonsils  are 
sometimes  also  included  under  this  name. 


Fig.   12. — Bronchial    casts    as    seen    when    carefully    spread    out    and 
viewed  over  a  black  background.     Natural  size. 

5.  Bronchial  Casts. — These  are  branching,  tree-like 
casts  of  the  bronchi,  frequently,  but  not  always,  com- 
posed of  fibrin  (Fig.  12).  In  color  they  are  usually 
white  or  grayish,  but  may  be  reddish  or  brown,  from 
the  presence  of  blood-pigment.     Their  size  varies  with 


62  THE    SPUTUM 

that  of  the  bronchi  in  which  they  are  formed.  Casts 
15  or  more  centimeters  in  length  have  been  observed, 
but  they  are  usually  very  much  smaller.  Ordinarily 
they  are  coiled  into  a  ball  or  tangled  mass  and  can  be 
recognized  only  by  floating  out  in  water — best  over  a 
black  background — when  their  tree-Uke  structure  be- 
comes evident.  The  naked-eye  examination  will  usu- 
ally suffice;  occasionally  a  hand  lens  may  be  required. 
Bronchial  casts  appear  in  the  sputum  in  croupous 
pneumonia,  in  fibrinous  bronchitis,  and  in  diphtheria 
when  the  process  extends  into  the  bronchi.  In  diph- 
theria they  are  usually  large.  In  fibrinous  or  chronic 
plastic  bronchitis  they  are  of  medium  size  and  usually 
of  characteristic  structure.  Their  demonstration  is 
essential  for  the  diagnosis  of  this  disease.  In  some 
cases  they  may  be  found  every  day  for  considerable 
periods;  in  others,  only  occasionally.  In  almost 
every  case  of  croupous  pneumonia  the  casts  are  pres- 
ent in  the  sputum  in  variable  numbers  during  the 
stage  of  hepatization  and  beginning  resolution.  Here 
they  are  usually  small  (0.5  to  i  cm.  in  length)  and  are 
often  not  branched. 

II.  MICROSCOPIC  EXAMINATION 

The  portions  most  likely  to  contain  structures  of 
interest  should  be  very  carefully  selected,  as  already 
described.  The  Jew  minutes  spent  in  this  preliminary 
examinatian  will  sometimes  save  hours  of  work  later. 
Opaque,  white  or  yellow  particles  are  most  frequently 
bits  of  food,  but  may  be  cheesy  masses  from  the  tonsils; 
small  cheesy  nodules,  derived  from  tuberculous  cavities 
and  containing  many  tubercle  bacilli  and  elastic  fibers; 


MICROSCOPIC   EXAMINATION  63 

Curschmann's  spirals,  or  small  fibrinous  casts,  coiled 
into  little  balls;  or  shreds  of  mucus  with  great  numbers 
of  entangled  pus-corpuscles.  The  food-particles  most 
apt  to  cause  confusion  are  bits  of  bread,  which  can  be 
recognized  by  the  blue  color  which  they  assume  when 
touched  with  iodin  solution. 

Some  structures  are  best  identified  without  staining; 
others  require  that  the  sputum  be  stained. 

A.  Unstained  Sputum 

A  careful  study  of  the  unstained  sputum  should  be 
included  in  every  routine  examination.  Unfortunately 
it  is  almost  universally  neglected.  It  best  reveals  cer- 
tain structures  which  are  seen  imperfectly  or  not  at  all 
in  stained  preparations.  It  gives  a  general  idea  of  the 
other  structures  which  are  present,  such  as  pus-cor- 
puscles, eosinophiles,  epithelial  cells,  and  blood  and 
thus  suggests  appropriate  stains  to  be  used  later.  It 
enables  one  to  select  more  intelhgently  the  portions  to 
be  examined  for  tubercle  bacilli. 

The  particle  selected  for  examination  should  be  trans- 
ferred to  a  clean  slide,  covered  with  a  clean  cover-glass, 
and  examined  with  the  i6-mm.  objective,  followed  by 
the  4  mm.  The  oil-immersion  lens  should  not  be  used 
for  this  purpose.  It  is  convenient  to  handle  the  bits 
of  sputum  with  a  wooden  tooth-pick  or  with  a  wooden 
cotton-applicator,  which  may  be  burned  when  done 
with.  The  platinum  wire  used  in  bacteriologic  work 
is  unsatisfactory  because  not  usually  stiff  enough.  A 
little  practice  is  necessary  before  one  can  handle 
particles  of  sputum  readily.  The  bit  desired  should 
be  separated  from  the  bulk  of  the  sputum  by  cutting 


64  THE   SPUTUM 

it  free  with  the  toothpick  and  drawing  it  out  upon  the 
dry  portion  of  the  glass  dish.  It  can  then  be  picked 
up  by  rotating  the  end  of  a  fresh  tooth-pick  against  it. 
T}ie  slide  must  never  he  dipped  into  the  sputum,  nor  must 
any  of  the  sputum  he  allowed  to  reach  its  edges  in 
spreading. 

The  more  important  structures  to  be  seen  in  unstained 
sputum  are:  elastic  fibers,  Curschmann's  spirals,  Char- 
cot-Leyden  crystals,  pigmented  cells,  myelin  globules, 
the  ray  fungus  of  actinomycosis,  and  molds.  Forming 
the  background  for  these  are  usually  pus-corpuscles, 
granular  detritus,  and  mucus  in  the  form  of  translucent, 
finely  fibrillar  or  jelly-like  masses.  The  pus  cells  ap- 
pear as  finely  granular  grayish  or  yellowish  balls  about 
12  /i  in  diameter  and  without  visible  nuclei  (see  Figs. 
20,  21).     They  are  best  studied  in  stained  preparations. 

1.  Elastic  Fibers. ^ — These  are  the  elastic  fibers  of 
the  pulmonary  substance,'  where  they  are  distributed  in 
the  walls  of  the  alveoli,  the  bronchioles,  and  the  blood- 
vessels. When  found  in  the  sputum  they  always 
indicate  destructive  disease  of  the  lung,  provided  they 
do  not  come  from  the  food,  which  is  a  not  infrequent 
source.  They  are  found  most  commonly  in  phthisis; 
rarely  in  other  diseases.  Advanced  cases  of  tuberculosis 
often  show  great  numbers,  and,  rarely,  they  may  be 
found  in  early  tuberculosis  when  the  bacilli  cannot  be 
detected.  After  the  diagnosis  is  established  they  fur- 
nish a  valuable  clue  as  to  the  existence  and  rate  of  lung 
destruction.  In  gangrene  of  the  lung,  contrary  to  the 
older  teaching,  elastic  tissue  is  probably  always  present 
in  the  sputum,  usually  in  large  fragments. 

The  fibers  should  be  searched  for  with  a  i6-mm.  ob- 


MICROSCOPIC  EXAMINATION 


65 


jective,  although  a  higher  power  is  needed  to  identify 
them  with  certainty.  They  may  usually  be  more  clearly 
seen  if  a  drop  of  10  to  20  per  cent,  caustic  soda  solu- 


FiG.   13. — Elastic  fibers  in  tuberculous  sputum,  unstained,  as  seen  with 
a  low  power  objective  (  X  lOo). 


Fig.  14. — Elastic   fibers  and   pus-corpuscles  in   tuberculous   sputum, 
unstained  (  X  300). 

tion  be  mixed  with  the  sputum  on  the  slide  before  the 
cover  is  applied.  Under  the  4  mm.  they  appear  as 
slender,  highly  refractive,  wavy  libers  with  double  con- 
tour, and  often  curled  or  split  ends.     Frequently  they 


66  THE   SPUTUM 

are  found  in  alveolar  arrangement,  retaining  the  original 
outline  of  the  alveoli  of  the  lung  (Figs.  13  and  14). 
This  arrangement  is  positive  proof  of  their  origin  in  the 
lung. 

Leptothrix  buccalis,  which  is  a  normal  inhabitant  of  the 
mouth,  may  easily  be  mistaken  for  elastic  tissue.  It  can 
be  distinguished  by  running  a  little  Lugol's  solution 
under  the  cover-glass  (see  p.  535).  Fatty-acid  crystals, 
which  are  often  present  in  Dittrich's  plugs  and  in  spu- 
tum which  has  lain  in  the  body  for  some  time,  also 
simulate  elastic  tissue  when  very  long,  but  they  are  more 
like  stiff,  straight  or  curved  needles  than  wavy  threads. 
They  show  varicosities  when  the  cover-glass  is  pressed 
upon.  The  structures  which  most  frequently  confuse 
the  student  are  the  cotton  fibrils  which  are  present  as  a 
contamination  in  most  sputa.  These  are  usually 
coarser  than  elastic  fibers,  and  flat,  with  one  or  two 
twists,  and  often  have  longitudinal  striations  and  frayed- 
out  ends.  In  stained  preparations  students  frequently 
report  the  fibrils  of  precipitated  mucus  as  elastic 
tissue. 

Elastic  fibers  from  the  food  are  coarser,  less  frequently 
wavy  and  not  arranged  in  alveolar  order. 

To  find  elastic  fibers  when  not  abundant,  boil  the 
sputum  with  a  10  per  cent,  solution  of  caustic  soda  until 
it  becomes  fluid ;  add  several  times  its  bulk  of  water,  and 
centrifugalize,  or  allow  to  stand  for  twenty-four  hours 
in  a  conical  glass.  Examine  the  sediment  microscopic- 
ally. The  fibers  will  be  pale  and  swollen  and,  therefore, 
somewhat  difficult  to  recognize.  Too  long  boiling  will 
destroy  them  entirely. 

The  above  procedure,  although  widely  recommended, 


MICROSCOPIC   EXAMINATION  67 

will  rarely  or  never  be  necessary  if  the  sputum  is  care- 
fully examined  in  a  thin  layer  against  a  black  back- 
ground macroscopically  and  with  a  hand-lens,  and  if 
all  suspicious  portions  are  further  studied  with  the 
microscope. 

2.  Curschmann's  Spirals.' — These  peculiar  struc- 
tures are  found  most  frequently  in  bronchial  asthma,  of 
which  they  are  fairly  characteristic.     Although  not  pres- 


FiG.  15. — Curschmann's  spirals  in  asthmatic  sputum  as  seen  when 
pressed  out  between  two  plates  of  glass  and  viewed  over  a  black  back- 
ground.    Each  is  embedded  in  a  mass  of  grayish  mucus.     Natural  size 

ent  in  every  attack,  they  probably  occur  at  some  time 
in  every  case.  Sometimes  they  can  be  found  only  near 
the  end  of  the  attack.  They  may  occasionally  be  met 
with  in  chronic  bronchitis  and  other  conditions.  Their 
nature  has  not  been  definitely  determined. 

Macroscopically,  they  are  whitish  or  yellow,  wavy 
threads,  frequently  coiled  into  little  balls  (Fig.  15). 
Their  length  is  rarely  over  1.5  cm.,  though  it  some- 
times exceeds  5  cm.  They  can  sometimes  be  definitely 
recognized  with  the  naked  eye.  Under  a  i6-mm. 
objective  they  appear  as  mucous  threads  with  a  bright, 
colorless    central    line — the    so-called    central    fiber — 


^68 


THE    SPUTUM 


about  which  are  wound  many  fine  fibrils  (Figs.  i6  and 
17).  The  spiral  fibrils  are  sometimes  loosely,  some- 
times tightly  wound.     Eosinophiles  are  usually  present 


.^?' 


-#  4 


%y-:Ui 


Fig.   16. — End  of  a  large,  tightly  wound  Curschmanii  a  a^^iicn  in  sputum 
from  a  case  of  bronchial  asthma.     Unstained  (X  70). 


1  io.  i  7. — Slender,  loosely  wound  Curschmann's  spiral  in  sputum  from 
a  case  of  bronchial  asthma.  A  few  Charcot-Leyden  crystals  are  also 
shown.     Unstained  (X  70). 

within  them,  and  sometimes  Charcot-Leyden  crys- 
tals, also.  Not  infrequently  the  spirals  are  imperfectly 
formed,  consisting  merely  of  twisted  strands  of  mucus 


MICROSCOPIC   EXAMINATION  69 

enclosing  leukocytes.     The  central  fiber  is  absent  from 
these. 

3.  Charcot=Leyden  Crystals.  -  Of  the  crystals 
which  may  be  found  in  the  sputum,  the  most  interesting 
are  the  Charcot-Leyden  crystals.  They  may  be  absent 
when  the  sputum  is  expectorated,  and  appear  in  large 
numbers  after  it  has  stood  for  some  time.  They  are 
rarely  found  except  in  cases  of  bronchial  asthma,  and 


Fig.  18. — Charcot-Leyden  crystals  and  eosinophilic  leukocytes  in 
sputum  from  a  case  of  bronchial  asthma.  Unstained  ( X  475).  The  mag- 
nification is  greater  than  is  usually  used  in  studying  these  structures. 

were  at  one  time  thought  to  be  the  cause  of  the  disease. 
They  frequently  adhere  to  Curschmann  spirals.  Their 
exact  nature  is  unknown.  Their  formation  seems  to  be 
in  some  way  connected  with  the  presence  of  eosinophilic 
cells.  Outside  of  the  sputum  they  are  found  in  the 
feces  in  association  with  animal  parasites,  and  in  the 
coagulated  blood  in  leukemia. 

They  are  colorless,  pointed,  often  needle-like  crystals 
(Fig.  18).  Formerly  they  were  described  as  octahedral, 
but  are  now  known  to  be  hexagonal  in  cross-section. 


7©  THE   SPUTUM 

Their  size  varies  greatly,  the  average  length  being  about 
three  or  four  times  the  diameter  of  a  red  blood-corpuscle. 

Other  crystals — hematoidin,  cholesterin,  and,  most 
frequently,  fatty-acid  needles  (see  Fig.  49) — are  com- 
mon in  sputum  which  has  remained  in  the  body  for  a 
considerable  time,  as  in  abscess  of  the  lung  and  bron- 
chiectasis. The  fatty-acid  crystals  are  regularly  found 
in  Dittrich's  plugs.  They  might  be  mistaken  for 
elastic  fibers  (see  p.  66).  Sometimes  they  form 
rounded  masses  with  the  individual  crystals  radially 
arranged  and  they  then  bear  considerable  resemblance 
to  the  clumps  of  Actinomyces  bovis. 

4.  Pigmented  Cells. — Granules  of  pigment  are 
sometimes  seen  in  ordinary  pus-corpuscles  but  the  more 
common  pigment-containing  cells  are  large  mononuclear 
cells  whose  origin  is  in  some  doubt.  They  were  for- 
merly thought  to  be  the  flattened  epithelial  cells  which 
line  the  pulmonary  alveoh.  The  present  tendency  is 
to  identify  them  with  the  large  mononuclear  leuko- 
cytes which  are  known  to  take  up  pigment  granules 
readily.  Two  kinds  of  pigmented  cells  deserve  men- 
tion: those  which  contain  blood-pigment,  chiefly  hemo- 
siderin; and  those  which  contain  carbon. 

To  those  which  contain  blood-pigment  the  name  heart 
failure  cells  has  been  given,  because  they  are  most 
frequently  found  in  long-continued  passive  congestion 
of  the  lungs  resulting  from  poorly  compensated  heart 
disease.  The  presence  of  these  cells  in  considerable 
numbers,  by  directing  one's  attention  to  the  heart, 
will  sometimes  clear  up  the  etiology  of  a  chronic  bron- 
chitis. They  are  sometimes  so  numerous  as  to  give  the 
sputum  a  brownish  tinge.     Such  cells  are  also  found 


MICROSCOPIC   EXAMINATION  7 1 

in  the  sputum  in  pulmonary  infarction  and  for  some 
time  after  a  pulmonary  hemorrhage.  In  fresh  un- 
stained sputum  heart  failure  cells  appear  as  round, 
grayish  or  colorless  bodies  filled  with  variously  sized 
rounded  granules  of  yellow  to  brown  pigment  (see 
Plate  II,  Fig.  i).  Sometimes  the  pigmentation  takes 
the  form  of  a  diffuse  staining.  The  nucleus  is  usually 
obscured  by  the  pigment.  The  cells  are  large,  aver- 
aging about  35  M  in  diameter. 

To  demonstrate  the  nature  of  the  brown  pigment  apply 
a  10  per  cent,  solution  of  potassium  ferrocyanid  for  a  few 
minutes  and  follow  with  weak  hydrochloric  acid.  Iron- 
containing  pigment  assumes  a  blue  color.  Many  of  the 
■granules,  will,  however,  fail  to  respond.  The  test  may  be 
applied  either  to  wet  preparations  or  to  dried  smears. 

Carbon-laden  cells  are  less  important  (see  Plate 
II,  Fig.  i).  They  are  especially  abundant  in  the  spu- 
tum of  anthracosis  where  angular  black  granules,  both 
intracellular  and  extracellular,  may  be  so  numerous 
as  to  color  the  sputum.  Similar  cells  with  smaller 
carbon  particles  are  often  abundant  in  the  morning 
sputum  of  those  who  inhale  tobacco  smoke  to  excess. 

5.  Myelin  Globules. — These  have  little  or  no  clin- 
ical significance  but  require  mention  because  of  the 
danger  of  confusing  them  with  more  important  struc- 
tures, notably  blastomyces.  They  are  colorless,  round 
or  oval  globules  of  various  sizes,  often  resembling  fat- 
droplets  but  more  frequently  showing  peculiar  con- 
centric or  irregularly  spiral  markings  (Figs.  19  and  27). 
Such  globules  are  abundant  in  the  scanty  morning 
sputum  of  apparently  healthy  persons,  but  may  be 


72  THE    SPUTUM 

found  in  any  mucoid  sputum.  They  lie  both  free  in 
the  sputum  and  contained  within  the  large  cells  which 
have  long  been  known  as  alveolar  cells  but  which  are 
possibly  larizc  mononuclear  leukocytes. 


'^ 


•a 


o. 


Fig.   19. —  M>c.  ;:ee  and  contained  within  cells.      From  a 

"normai  morning  sputum"  (X  350). 

6.  Actinomyces  Bovis  (Ray=fungus). — In  the  spu- 
tum of  pulmonary  actinomycosis  and  in  the  pus  from 
actinomycotic  lesions  elsewhere,  small,  yellowish,  "sul- 
phur" granules  can  be  detected  with  the  unaided  eye. 
Without  a  careful  macroscopic  examination  they  are 
almost  certain  to  be  overlooked.  The  fungus  can  be 
seen  by  crushing  one  of  these  granules  between  slide 
and  cover,  and  examining  with  a  low  power.  It 
consists  of  a  network  of  threads  having  a  more  or  less 
radial  arrangement  (Figs  20  and  21).  In  cattle,  and 
to  a  less  extent  in  man,  the  filaments  at  the  periphery 
of  the  nodule  present  club-shaped  extremities.  It  can 
be  brought  out  more  clearly  by  running  a  little  solution 
of  eosin  in  alcohol  and  glycerin  under  the  cover.  This 
organism,  also  called  Streptothrix  actinomyces,  appar- 


MICROSCOPIC   EXAMINATION  73 

ently  stands  midway  between  the  bacteria   and   the 
molds.     It  stains  by  Gram's  method. 

Actinomycosis  of  the  lung  is  rare.     The  clinical  pic- 
ture is  that  of  tuberculosis. 


Fig.  20. — A  "sulphur  granule"  crublicd  'ueucHiii  the  cover  glass. 
From  the  pus  of  a  case  of  actinomycosis  of  submaxillary  lymph  nodes. 
Unstained  (  X  60). 


Fig.   21. — -A  portion  of  Fig.  20,  more  highly  magnified  (  X  300.) 

7.  Molds  and  Yeasts. — The  hyphse  and  spores  of 
various  molds  are  occasionally  met  with  in  the  sputum. 
They  are  usually  the  result  of  contamination,  and  have 
httle  significance.  The  hyphas  are  rods,  usually  jointed 
or  branched  (see  Fig.  79)  and  often  arranged  in  a  mesh- 


74      •  THE    SPUTUM 

work  (mycelium);  the  spores  are  highly  refractive 
spheres  and  ovoids.  Both  stain  well  with  the  ordinary 
stains. 

In  the  extremely  rare  condition  of  systemic  blasto- 
mycosis the  specific  yeasts  have  been  found  in  the  spu- 
tum in  large  numbers.  It  is  advisable  to  add  a  little 
lo  per  cent,  caustic  soda  solution  and  examine 
unstained. 

8.  Animal  Parasites. — These  are  extremely  rare  in 
the  sputum  in  this  country.  A  trichomonad,  perhaps 
identical  with  Trichomonas  intestinalis  has  been  seen  in 
the  sputum  of  putrid  bronchitis  and  gangrene  of  the 
lung,  but  its  causal  relationship  is  doubtful.  In  Japan, 
infection  with  the  lung  fluke-worm,  Paragonimus  wes- 
termannii,  is  common,  and  the  ova  are  found  in  the  spu- 
tum. The  lung  is  not  an  uncommon  seat  for  echino- 
coccus  cysts,  and  booklets  and  scolices  may  appear,  as 
may  also  Endamceha  histolytica,  when  a  hepatic  abscess 
.  has  ruptured  into  the  lung.  Larvae  of  Strongyloides  in- 
testinalis and  of  the  hook-worm  have  been  reported. 
Ciliated  body-cells,  with  cilia  in  active  motion,  are  not 
infrequently  seen,  and  may  easily  be  mistaken  for  infu- 
soria. All  the  above-mentioned  parasites  are  described 
in  Chapter  VI. 

B.  Stained  Sputum 

Structures  which  are  best  seen  in  stained  sputum  are 
bacteria  and  cells. 

A  number  of  smears  should  be  made  upon  slides  or 
covers.  These  films  must,  of  course,  be  thin,  but  it  is 
easily  possible  to  get  them  too  thin.  This  is  a  common 
error  of  students  who  have  just  finished  a  course  in 


MICROSCOPIC   EXAMINATION  75 

bacteriology  and  who  have  there  been  accustomed  to 
work  with  scarcely  perceptible  films  of  bacteria.  It  is 
a  good  plan  to  slide  ofi  the  cover-glass  from  the  prepara- 
tion used  for  the  unstained  microscopic  examination. 
If  this  is  properly  done  satisfactory  smears  will  be  left 
on  both  slide  and  cover.  They  are  then  dried  in  the  air, 
and  fixed  in  the  flame,  as  described  on  page  5  7 1 ,  or  better, 
by  immersion  for  one  or  two  minutes  in  pure  wood  alco- 
hol or  saturated  solution  of  corrosive  sublimate.  Fixa- 
tion will  ordinarily  kill  the  bacteria  and  the  smears  may 
be  kept  indefinitely;  but  smeats  on  slides  when  fixed  by 
heat  are  often  not  sterile,  and  should  be  handled  ac- 
cordingly. One  of  the  smears  should  be  stained  with 
some  general  stain,  like  Loffler's  methylene  blue  or  py- 
ronin-methyl  green  (see  p.  642),  which  will  give  a  good 
idea  of  the  various  cells  and  bacteria  present.  Special 
stains  may  then  be  applied,  as  indicated,  but  a  routine 
examination  should,  in  all  cases,  include  a  stain  by 
the  method  for  the  tubercle  bacillus  and  by  Gram's 
method. 

1 .  Bacteria. — Saprophytic  bacteria  from  mouth  con- 
tamination are  frequently  present  in  large  numbers  and 
will  prove  confusing  to  the  inexperienced.  The  pres- 
ence of  squamous  cells  in  their  neighborhood  wiU  sug- 
gest their  source.  Among  the  pathogenic  organisms 
are:  tubercle  bacilli;  staphylococci  and  streptococci; 
pneumococci;  bacilli  of  Friedlander;  influenza  bacilli; 
and  Micrococcus  catarrhalis.  Of  these  the  tubercle 
bacillus  is  the  "only  one  whose  recognition  has  great 
clinical  value  and  the  only  one  which  is  easily  identified 
in  stained  smears.  Their  cultural  characteristics  are 
described  in  Chapter  VIII. 


76  THE    SPUTUM 

(i.)  Tubercle  Bacillus. — The  presence  of  tubercle  ba- 
cilli may  be  taken  as  positive  evidence  of  the  existence 
of  tuberculosis  somewhere  along  the  respiratory  tract, 
most  likely  in  the  lung.  In  laryngeal  tuberculosis  they 
are  not  easily  found  in  the  sputum,  but  can  frequently 
be  detected  in  swabs  made  directly  from  the  larynx. 

The  importance  of  carefully  selecting  the  portion  for 
examination  cannot  be  too  strongly  emphasized.  It 
is  always  best  to  select  the  more  purulent  portions  of 
the  sputum,  keeping  away  from  the  mucoid  parts.  If 
bits  of  necrotic  tissue  are  present  they  may  show  im- 
mense numbers  of  tubercle  bacilli,  when  other  portions 
of  the  specimen  contain  very  few.  One  must,  however, 
be  on  his  guard  against  bits  of  food  which  resemble 
these  "caseous  particles."  The  specimen  should  be 
examined  while  fresh.  It  wUl  usually  liquefy  upon 
standing,  and  this,  by  preventing  the  selection  of  parti- 
cles favorable  for  examination,  will  greatly  reduce  one's 
chances  of  finding  bacilli. 

Recognition  of  the  tubercle  bacillus  depends  upon  the 
fact  that  it  stains  with'  difficulty;  but  that  when  once 
stained,  it  retains  the  stain  tenaciously,  even  when 
treated  with  a  mineral  acid,  which  quickly  removes  the 
stain  from  other  bacteria.  This  "acid-fast "  property  is 
due  to  the  presence  of  a  waxy  or  lipoid  substance.  A 
number  of  the  best  staining  methods  are  included  here. 
Since  Gabbet's  method  is  convenient,  inexpensive  and 
widely  used  in  oflace  work,  it  is  given  in  greater  detail 
than  the  others.  The  author,  however,  would  recom- 
mend Pappenheim's  method  for  routine  work,  as  least 
likely  to  give  trouble  to  the  inexperienced.  Students 
rarely  fail  to  get  perfect  results  at  the  first  trial. 


MICROSCOPIC    EXAMINATION  ^77 

Tubercle  bacilli  can  often  be  found  in  very  poorly 
prepared  slides,  but  for  dependable  results  when  bacilli 
are  scarce  properly  spread,  fixed,  and  stained  prepara- 
tions free  from  precipitated  stain  are  absolutely  essen- 
tial. The  person  who  is  content  with  an  imperfect 
preparation  because  it  is  "good  enough  for  diagnosis" 
will  succeed  only  in  the  most  obvious  cases. 

Gabbet's  Method. — i.  Spread  suspicious  particles  thinly 
and  evenly  upon  a  slide  or  a  cover-glass  held  in  the  grasp  of 
cover-glass  forceps.  In  general,  slides  are  more  satisfac- 
tory, but  cover-glasses  are  easier  to  handle  while  staining. 
Do  not  grasp  a  cover  too  near  the  edge  or  the  stain  will  not 
stay  on  it  well.  Tenacious  sputum  will  spread  better  if 
gently  warmed  while  spreading. 

2.  Dry  the  film  in  the  air. 

3.  Fix  the  film  by  immersing  in  saturated  aqueous  solu- 
tion of  corrosive  sublimate  or  in  methyl  alcohol  for  two  or 
three  minutes,  and  then  rinse  well  in  water.  This  is  much 
to  be  preferred,  particularly  for  beginners,  to  the  usual  prac- 
tice of  fixing  in  the  flame  (see  p.  572).  Should  the  film  be 
washed  off  during  future  manipulations,  fixation  has  been 
insufficient. 

4.  Apply  as  much  carbol-fuchsin  (see  p.  639)  as  will  stay 
on,  and  hold  over  a  flame  so  that  it  will  steam  for  three 
minutes  or  longer,  replacing  the  stain  with  a  dropper  as  it 
evaporates.  If  the  stain  is  allowed  to  evaporate  com- 
pletely, the  preparation  is  ruined.  If  the  bacilli  are  well 
stained  in  this  step,  there  will  be  little  danger  of  decoloriz- 
ing them  later.  Too  great  heat  will  interfere  with  the 
staining  of  some  of  the  bacilli,  probably  by  destroying  the 
waxy  substance  upon  which  the  acidfast  property  depends. 

Recently  it  has  been  shown  that  fifteen  to  twenty  minutes 
staining  at  room  temperature  will  suffice,  and  this  may  be 


78  THE   SPUTUM 

recommended  on  the  score  of  avoiding  precipitates.  With 
most  batches  of  carbol-fuchsin  even  five  minute's  staining 
is  suflScient. 

5.  Wash  the  film  in  water. 

6.  Apply  Gabbet's  stain  (see  p.  641.)  to  the  under  side 
of  the  cover-glass  to  remove  excess  of  carbol-fuchsin,  and 
then  to  the  film-side.  Allow  this  to  act  for  one-fourth 
to  one-half  minute. 

7.  Wash  in  water. 

8.  If,  now,  the  thinner  portions  of  the  film  are  blue,  pro- 
ceed to  the  next  step;  if  they  are  still  red,  repeat  steps  6 
and  7  until  the  red  has  disappeared.  Too  long  application 
of  Gabbet's  stain  will  decolorize  the  tubercle  bacilH. 

9.  Place  the  preparation  between  layers  of  filter-paper 
and  dry  by  rubbing  with  the  fingers,  as  one  would  in  blotting 
ink.     Warm  over  the  flame  until  thoroughly  dry. 

10.  Put  a  drop  of  Canada  balsam  upon  a  clean  slide,  place 
the  cover-glass  film  side  down  upon  it,  and  examine  with  an 
immersion  objective.  Cedar  oil  or  water  may  be  used  in 
place  of  balsam  for  temporary  preparations.  Smears  on 
slides  may  be  examined  directly  with  an  oil-immersion  lens, 
no  cover  being  necessary. 

Ziehl-Neelson  Method. — The  objection  is  often  made  to 
the  above  method  that  decolorization  is  masked  by  the  blue 
in  Gabbet's  stain.  Although  this  will  not  make  trouble  if 
step  8  is  carefully  carried  out,  most  experienced  workers 
prefer  the  Ziehl-Neelson  method.  This  resembles  Gabbet's 
method,  with  the  following  exceptions:  After  the  staining 
with  carbol-fuchsin  the  smear  is  washed  in  5  per  cent,  nitric 
acid  (or,  better,  a  mixture  of  3  c.c.  concentrated  hydrochloric 
acid  and  97  c.c.  70  per  cent,  alcohol)  until  decolorized, 
washed  in  water,  stained  lightly  with  Lofller's  methylene 
blue,  again  washed,  and  finally  dried  and  mounted. 

Pappenheim's  Method. — This  is  the  same  as  Gabbet's 


PLATE  II 


w 

1 

1 

f» 

*5^ 

•"^V 

■  •«  '• 

<v^ 

0 

-'.>i 

.-i^- 

I. 

1'': 

i* 

'.""'. 

* 

Fig.  1. — Heart-failure  cells  and  car- 
bon-laden cells  in  unstained  sputum. 
Two  small  squamous  epitiielial  cells  and 
four  red  blood-corpuscles  are  included  for 
comparison  of  size.     X  zoo- 


Fig.  2. — Eosinophilic  leukocytes  and 
staphylococci  in  asthmatic  sputum.  Eo- 
sin  and  methylene-blue.     X  looo. 


\ 

>  if  I 

1      ! 

r 

\ 

-^ 

1* 

)  ^ 

-a 

^^TJH 

1 

r-;«^v.^ 

••• 

• 

..%. 

Fig.  3. — Tubercle  bacilli,  streptococci, 
pus  corpuscles,  and  mucous  threads  in 
tuberculous  sputum.  Ziehl-Neelson 
method.     X  1000. 


Fig.  4. — Much's  granules  from  two 
fields  of  a  slide  stained  as  described  in 
the  text.  A  group  of  half-digested  staphy- 
lococci is  also  shown.     X  isoo. 


MICROSCOPIC   EXAMINATION  79 

method,  except  that  Pappenheim's  methylene-blue  solution 
(see  p.  641)  is  substituted  for  Gabbet's  stain. 

The  method  is  very  satisfactory  for  routine  work,  De- 
colorization  of  the  tubercle  bacillus  is  practically  impos- 
sible: it  retains  its  red  color,  even  when  soaked  overnight 
in  Pappenheim's  solution.  The  stain  was  originally  recom- 
mended as  a  means  of  differentiating  the  smegma  bacillus, 
which  is  decolorized  by  it;  but  it  is  not  to  be  absolutely  relied 
upon  for  this  purpose. 

In  films  stained  by  these  methods  tubercle  bacilli, 
if  present,  will  be  seen  as  slender  red  rods  upon  a  blue 
background  of  mucus  (appearing  as  delicate  threads  and 
strands),  granular  detritus,  and  cells  (Plate  II,  Fig.  3). 
They  vary  considerably  in  size,  averaging  3  to  4  /i  in 
length — about  one-half  the  diameter  of  a  red  blood-cor- 
puscle. Beginners  must  be  warned  against  mistaking 
the  edges  of  cells,  or  particles  which  have  retained  the 
red  stain,  for  bacilli.  The  appearance  of  the  bacilli  is 
almost  always  typical,  and  if  there  seems  room  for 
doubt,  the  structure  in  question  is  probably  not  a  tu- 
bercle bacillus.  They  may  lie  singly  or  in  groups. 
They  are  very  frequently  bent  and  often  have  a  beaded 
appearance.  It  is  possible  that  the  larger,  beaded 
bacilli  indicate  a  less  active  tuberculous  process  than  do 
the  smaller,  uniformly  stained  ones.  Sometimes  they 
are  present  in  great  numbers — thousands  in  a  field  of 
the  2-mm.  objective,  Sometimes,  even  in  advanced 
cases,  several  cover-glasses  must  be  examined  to  find 
a  single  bacillus.  At  times  they  are  so  few  that  none 
are  found  in  stained  smears,  and  special  methods  are 
required  to  detect  them.  The  number  may  bear  some 
relation  tp  the  severity  of  the  disease,  but  this  relation 


8o  THE    SPUTUM 

is  by  no  means  constant.  The  mucoid  sputum,  from 
an  incipient  case  sometimes  contains  great  numbers, 
while  sputum  from  large  tuberculous  cavities  at  times 
contains  very  few.  Failure  to  find  them  is  not  conclu- 
sive, though  their  absence  is  much  more  significant 
when  the  spukim  is  purulent  than  when  it  is  mucoid. 

When  it  is  desired  to  record  the  approximate  number 
of  bacilli  present,  the  Gaffky  table  as  modified  by  Brown 
may  be  employed,  using  an  oil-immersion  lens  and  4X 
ocular : 

I.  One  to  four  bacilli  to  the  slide. 

II.  Average  of  one  in  many  fields. 

III.  Average  of  one  in  a  field. 

IV.  Average  of  two  to  three  in  a  field. 
V.  Average  of  four  to  six  in  a  field. 

VI.  Average  of  seven  to  twelve  in  a  field. 

VII.  Average  of  thirteen  to  twenty-five  in  a  field. 

VIII.  Average  of  about  fifty  in  a  field. 

IX.  Average  of  about  one  hundred  in  a  field. 

X.  Enormous  numbers  in  a  field. 

Since  the  sputum  raised  at  various  times  in  the  day, 
and  even  different  parts  of  the  same  sample,  may  vary 
greatly  in  bacillary  content,  such  a  table  is  of  little  value 
unless  the  twenty-four-hour  sputum  is  collected  and  uni- 
formly mixed  before  preparing  the  slides. 

When  bacilli  are  not  found  in  suspected  cases,  one  of 
the  following  methods  should  be  tried: 

I.  Antiformin  Method. — This  has  lately  come  into  use, 
and  has  superseded  the  older  methods  of  concentration. 
The  chief  difficulty  with  the  older  methods,  such  as  boiUng 
with  caustic  soda,  is  that  the  bacilli  are  so  injured  in  the 
process  that  they  do  not  stain  characteristically. 


MICROSCOPIC   EXAMINATION  8 1 

Antiformin  is  a  trade  name  for  a  preparation  consisting 
essentially  of  equal  parts  of  a  15  per  cent,  solution  of  caustic 
soda  and  a  20  per  cent,  solution  of  sodium  hypochlorite.  It 
keeps  fairly  well.  Substitutes  appear  to  be  less  satisfactory 
than  the  original  preparation. 

Loffler's  method  is  probably  the  best  for  clinical  work. 
It  kills  the  bacilli,  so  that  there  is  no  danger  in  handling 
the  material.  Upon  this  account,  however,  it  is  not  appli- 
cable to  isolation  of  tubercle  bacilli  for  cultures. 

Place  10  to  20  c.c.  of  the  sputum  in  a  small  flask,  with 
an  equal  amount  of  50  per  cent,  antiformin,  and  heat  to 
the  boiling-point.  The  sputum  will  be  thoroughly  lique- 
fied, usually  within  a  few  seconds.  For  each  10  c.c.  of  the 
resulting  fluid  add  1.5  c.c.  of  a  mixture  of  i  volume  of 
chloroform  and  9  volumes  of  alcohol.  Shake  vigorously  for 
several  minutes  or  until  emulsification  has  taken  place.  The 
object  is  to  impregnate  the  lipoid  capsule  of  the  bacilli  with 
chloroform,  thus  increasing  their  specific  gravity.  Pour  off 
the  emulsion  into  centrifuge  tubes  and  centrifugalize  at  high 
speed  for  about  fifteen  minutes.  The  chloroform  will  go  to 
the  bottom,  and  the  sediment  which  collects  on  its  surface 
in  a  thin  firm  layer  will  contain  the  tubercle  bacilli.  Pour 
off  the  supernatant  liquid  and  transfer  the  sediment  to  glass 
slides,  removing  the  excess  of  fluid  with  filter-paper.  To 
facilitate  removal  of  the  disk  of  sediment  in  toto  Williamson 
recommends  the  use  of  a  centrifuge  tube,  the  lower  ]/2  inch 
of  which  is  of  uniform  caliber  and  the  bottom  of  which  is 
open  and  plugged  with  a  rubber  stopper.  Add  a  little  egg- 
albumen  solution  (see  p.  87)  or,  better,  some  of  the  original 
sputum,  to  cause  the  film  to  adhere  to  the  slide,  mix  well, 
spread  into  a  uniform  layer,  and  finally  dry,  fix,  and  stain  by 
the  Ziehl-Neelson  method.  Lo filer  recommends  o.i  per 
cent,  solution  of  malachite  green  for  counterstain. 

2.  Animal  Inoculation. — Inoculation  of  guinea-pigs  is  the 
court  of  last  appeal  in  detection  of  tubercle  bacilli,  but  even 


82  THj:    SPUTUM 

this  is  not  infallible  for  it  has  been  shown  that  the  injected 
material  must  contain  lo  to  150  bacilli  in  order  to  produce 
tuberculosis  in  the  guinea-pig,  the  number  required  depend- 
ing upon  their  virulence.  The  method  is  described  on 
page  534- 

There  are  a  number  of  bacilli,  called  acid-fast  bacilli, 
which  stain  in  the  same  way  as  the  tubercle  bacillus. 
They  stain  with  difficulty,  and  w^hen  once  stained,  retain 
the  color  even  when  treated  with  a  mineral  acid;  but, 
unlike  the  tubercle  bacillus,  most  of  them  can  be  decolor- 
ized with  alcohol.  Of  these,  the  smegma  bacillus  is  the 
only  one  likely  ever  to  cause  confusion.  It,  or  a  similar 
bacillus,  is  sometimes  found  in  the  sputum  of  gangrene 
of  the  lung.  It  occurs  normally  about  the  glans  penis 
and  the  clitoris,  and  is  often  present  in  the  urine  and 
in  the  wax  of  the  ear.  The  method  of  distinguish- 
ing it  from .  the  tubercle  bacillus  is  given  later  (see 
p.  236). 

Other  bacteria  than  the  acid-fast  group  are  stained 
blue  by  Gabbet's  and  the  Ziehl-Neelson  method.  Those 
most  commonly  found  are  staphylococci,  streptococci, 
and  pneumococci.  Their  presence  in  company  with  the 
tubercle  bacillus  constitutes  mixed  infection,  which  is 
much  more  serious  than  single  infection  by  the  tubercle 
bacillus.  It  is  to  be  remembered,  however,  that  a  few 
of  the  bacteria  may  reach  the  sputum  from  the  upper 
air-passages  and  that  great  numbers  are  usually  present 
in  decomposing  sputum.  Clinically,  mixed  infection 
is  evidenced  by  fever. 

Within  the  past  few  years  much  interest  has  centered 
in  the  so-called  "Much's  granules."  These  are  Gram- 
positive    but    non-acid-fast    granules    which    are    ap- 


MICROSCOPIC   EXAMINATION  83 

parently  forms  of  the  tubercle  bacillus,  since  material 
containing  them  causes  tuberculosis  when  injected  into 
guinea-pigs.  They  may  be  present  either  alone  or  in 
company  with  the  ordinary  acid-fast  form. 

It  is  now  fairly  well  established  that  Much's  granules 
represent  a  less  virulent  form  of  the  tubercle  bacillus 
which  is  especially  frequent  in  quiescent  and  mild 
chronic  cases,  and  that  they  give  place  to  the  acid-fast 
forms  when  such  cases  become  active.  Their  detection 
is  therefore  important,  but  it  is  not  easy  because  of  other 
granules — precipitated  stain,  micrococci,  etc.— which 
may  be  mistaken  for  the  true  Much  bodies.  The  fol- 
lowing method,  while  somewhat  complicated,  reduces 
the  chance  for  error  to  the  minimum. 


M 

HHHB-' 

, 

1  1        iiipiyi 

"1 

;  .'vv. 

0 

■*   --'A''' 

m, 

1 

ijKw'              ..2z.:m 

Fig.   22. —  Much's  granules  in  sputum  stained  by  the  method  detailed 
in  the  text  (X  1500). 


Staining  Method  for  Much's  Granules. — i.  To  the 
twenty-four-hour  amount  of  sputum  add  an  equal  volume  of 
0.6  per  cent,  sodium  carbonate  solution,  shake  thoroughly, 
and  allow  to  stand  in  a  warm  place  (preferably  the  incu- 
bator) for  twenty-four  hours.  If  it  is  not  then  completely 
homogeneous  extend  the  time  to  forty-eight  hours. 


84  THE    SPUTUM 

2.  Centrifuge  thoroughly,  remove  half  of  the  supernatant 
fluid  and  mix  an  equal  volume  of  30  per  cent,  antiformin 
with  the  remaining  half.  Allow  this  to  act  for  twenty  min- 
utes.    It  is  imperative  that  the  antiformin  be  fresh. 

3.  Centrifugalize,  and  make  smears  from  the  sediment. 

4.  Stain  one  smear  by  the  Ziehl-Neelson  method.  This 
will  demonstrate  the  ordinary  tubercle  bacilli,  if  present, 
and  will  also  serve  to  show  whether  any  cocci  have  been  left 
undigested. 

5.  Immerse  the  remaining  smears  for  forty-eight  hours 
in  a  stain  consisting  of: 

Carbol-fuchsin 3  parts 

Carbol-methyl  violet i  part 

This  stain  remains  good  for  about  two  weeks.  The  car- 
bol-methyl violet  used  for  this  purpose  consists  of  2  per 
cent,  phenol,  9  parts;  saturated  alcoholic  solution  of  methyl 
violet,  I  part.  In  order  to  avoid  precipitates  smears  should 
stand  on  edge  while  in  the  stain. 

6.  Rinse  gently  in  water. 

7.  Cover  with  Gram's  iodin  solution  for  five  minutes, 
warming  until  steam  rises. 

8.  Decolorize  successively  with  5  per  cent,  nitric  acid  for 
one  minute,  with  3  per  cent,  hydrochloric  acid  for  ten  sec- 
onds, and  finally  with  a  mixture  of  equal  parts  of  acetone 
and  95  per  cent,  alcohol  until  color  ceases  to  come  off. 

9.  Dry,  mount  in  cedar  oil  or  balsam  and  examine  with 
the  oil-immersion  lens. 

Steps  I  and  2  in  this  method  serve  the  double  purpose 
of  concentrating  the  sputum  and  of  digesting  any  micro- 
cocci which  may  be  present  and  which  might  be  confused 
with  Much's  granules.  One  must  be  extremely  cautious 
in  interpreting  isolated  granules  if  any  undigested  cocci  are 
found  in  the  control  slide.     Partially  digested  cocci  which 


MICROSCOPIC   EXAMINATION  85 

take  the  color  of  the  background  will  not  cause  confusion. 
The  concentration  and  digestion  may  be  omitted  if  desired, 
but  results  are  then  much  less  dependable. 

Much's  granules  (Plate  II,  Fig.  4)  are  definite,  clean-cut, 
round  or  oval  bodies  about  0.5  /i  in  diameter.  They  are 
thus  about  half  the  diameter  of  a  staphylococcus.  Ordi- 
narily they  are  a  deep  purple,  often  with  a  tinge  of  red. 
They  may  lie  singly  or,  more  frequently,  in  rows  of  two  to 
five.  Connecting  the  granules  can  usually  be  seen  a  faint 
bluish  or  reddish  band  suggesting  the  body  of  a  bacillus  in 
or  on  which  the  granules  lie.  Isolated  granules  usually 
appear  to  lie  at  the  end  or  in  the  middle  of  such  a  band,  and 
unless  the  band  is  seen,  they  should  not  be  accepted  as  true 
Much's  granules. 

(2)  Staphylococcus  and  Streptococcus  (seep.  516). — 
One  or  both  of  these  organisms  is  commonly  present  in 
company  with  the  tubercle  bacillus  in  the  sputum  of 
advanced  phthisis  (Plate  II,  Figs.  2  and  3).  They  are 
often  found  in  bronchitis,  catarrhal  pneumonia,  and 
many  other  conditions.  The  streptococcus  is  a  common 
cause  of  severe  sore  throat  and  tonsillitis. 

(3)  Pneumococcus  (Dipiococcu^  of  Frankel). — The 
pneumococcus  is  the  causative  agent  in  nearly  all  cases 
of  croupous  pneumonia,  and  is  commonly  found  in  large 
numbers  in  the  rusty  sputum  of  this  disease.  It  is  fre- 
quently met  with  in  the  sputum  of  catarrhal  pneumonia, 
bronchitis,  and  tuberculosis.  It  is  also  an  important  fac- 
tor in  the  causation  of  pleurisy,  meningitis,  otitis  media, 
and  other  inflammations.  It  is  frequently  present  in 
the  saliva  in  health.  Pneumococci  are  about  the  size 
of  streptococci.  They  are  ovoid  in  shape,  and  lie  in 
pairs,  end  to  end,  often  forming  short  chains.     Each  is 


86  THE    SPUTUM 

surrounded  by  a  gelatinous  capsule,  which  is  its  dis- 
tinctive feature  (Fig.  23). 

The  pneumococcus  is  closely  related  to  the  strepto- 
coccus, and  it  is  sometimes  extremely  difficult  to  differ- 
entiate them  even  by  culture  methods  (for  which  see 
p.  581).  The  morphology  of  the  pneumococcus,  the 
fact  that  it  is  Gram-positive,  and  the  presence  of  a  cap- 
sule are,  however,  generally  sufficient  for  its  recognition 


Fig.  23. — Diplococcus  pn'eumonice  in  the  blood  (x  looo)  (Frankel  and 

Pfeiffer). 

in  smears  from  sputum  or  pus.  The  capsule  is  often 
seen  as  a  halo  around  pairs  of  cocci  in  smears  stained 
by  the  ordinary  methods,  particularly  Gram's  method, 
but  to  show  it  well  special  methods  are  required.  There 
are  numerous  special  methods  of  staining  capsules  which 
are  applicable  to  other  encapsulated  bacteria,  as  well  as 
to  the  pneumococcus,  but  few  of  them  are  satisfactory. 
Buerger's  method  can  be  recommended.  It  is  especially 
useful  with  cultures  upon  serum  media,  but  is  applicable 


MICROSCOPIC    EXAMINATION  87 

also  to  the  sputum.  Smith's  and  Rosenow's  methods 
are  easier  of  application,  and  apparently  give  uniformly 
good  results.  The  sputum  should  be  fresh — not  more 
than  three  or  four  hours  old. 

Buerger's  Method  for  Capsules. — i.  Mix  a  few  drops 
each  of  the  sputum  and  blood-serum  or  egg-albumen  solu- 
tion (egg-albumen,  distilled  water,  equal  parts;  shake,  filter 
through  cotton,  and  add  about  0.5  per  cent,  phenol).  Blood- 
serum  can  be  obtained  as  described  for  the  Widal  test  (see 
p.  606).  Make  thin  smears  from  the  mixture,  and  just  as 
the  edges  begin  to  dry,  cover  with  Midler's  fluid  (potassium 
dichromate,  2.5  Gm.;  sodium  sulphate,  i.o  Gm.;  water,  100 
c.c.)  saturated  with  mercuric  chlorid  (ordinarily  about  5 
per  cent.) .    Gently  warm  over  a  flame  for  about  three  seconds. 

2.  Rinse  very  quickly  in  water. 

3.  Flush  once  with  alcohol. 

4.  Apply  tincture  of  iodin  for  one  to  two  minutes. 

5.  Thoroughly  wash  off  the  iodin  with  alcohol  and  dry  in 
the  air. 

6.  Stain  about  three  seconds  with  weak  anilin-gentian 
violet  freshly  made  up  as  follows:  Anilin  oil,  10;  water,  100; 
shike;  filter;  and  add  5  c.c.  of  a  saturated  alcoholic  solution 
of  gentian  violet. 

7.  Rinse  off  the  stain  with  2  per  cent,  solution  of  sodium 
chlorid,  mount  in  this  solution,  and  examine  with  an  oil- 
immersion  objective. 

Buerger  suggests  a  very  useful  variation  as  follows:  After 
the  alcohol  wash  and  drying,  the  specimen  is  stained  by 
Gram's  method  (see  p.  572),  counterstained  with  aqueous 
solution  of  fuchsin,  washed,  and  mounted  in  water.  The 
pneumococcus  holds  the  purple  stain,  while  all  capsules  take 
the  pink  counterstain. 

W.  H.  Smith's  Method.^ — i.  Make  thin  smears  of  the 
sputum  or  other  material,  which  should  be  as  fresh  as  possible. 


88  THE    SPUTUM 

2.  Fix  in  the  flame  in  the  usual  manner. 

3.  Apply  a  10  per  cent,  aqueous  solution  of  phospho- 
molybdic  acid  (Merck)  for  four  to  five  seconds. 

4.  Rinse  in  water. 

5.  Apply  anilin-gentian  violet  (see  p.  640),  steaming 
gently  for  fifteen  to  thirty  seconds. 

6.  Rinse  in  water. 

7.  Apply  Gram's  iodin  solution,  steaming  gently  for 
fifteen  to  thirty  seconds. 

8.  Wash  in  95  per  cent,  alcohol  until  the  purple  color 
ceases  to  come  off. 

9.  Rinse  in  water. 

10.  Apply  a  6  per  cent,  aqueous  solution  of  eosin  (Grli- 
bler,  w.  g.),  and  gently  warm  for  one-half  to  one  minute. 

11.  Rinse  in  water. 

12.  Wash  in  absolute  alcohol. 

13.  Clear  in  xylol. 

14.  Mount  in  balsam. 

This  is  essentially  Gram's  method  (see  p.  572),  preceded 
by  treatment  with  phosphomolybdic  acid  and  followed  by 
eosin.  Gram-positive  bacteria  like  the  pneumococcus  are 
deep  purple;  capsules  are  pink  and  stand  out  clearly. 

When  the  method  is  applied  to  Gram-negative  bacteria, 
steps  five  to  nine  inclusive  are  omitted;  and  between  steps 
eleven  and  twelve  the  preparation  is  counterstained  with 
Lofiler's  methylene  blue,  gently  warming  for  fifteen  to 
thirty  seconds. 

Rosenow's  Method. — This  is  the  same  as  Smith's  with 
the  exception  that  a  10  per  cent,  solution  of  tannic  acid, 
applied  while  the  film  is  still  moist  and  allowed  to  act 
for  ten  to  twenty  seconds  takes  the  place  of  the  heat  and 
phosphomolybdic  acid  in  steps  2  and  3. 

(4)  Bacillus  of  Friedlander  (Bacillus  mucosus  cap- 
sulatus). — In  a  small  percentage  of  cases  of  pneumonia 


MICROSCOPIC   EXAMINATION  89 

this  organism  is  found  alone  or  in  company  with  the 
pneumococcus.     Its  pathologic   significance  is  uncer- 
tain.    It  is  often  present  in  the  .„*skP'*t^ 
respiratory    tract    under    normal 
conditions.      Friedlander's  bacilli  \ 
are  non-motile,  encapsulated  rods,   .  '  ; 
sometimes      arranged     in     short   \    " 
chains  (Fig.  24) .     Very  short  indi- 
viduals in   pairs  closely  resemble 


pneumococci,  from  which  they  are     ^^^-   24— Friediand- 

^,,  'ii  1  1         ^^^  bacillus  in  pus  from 

distinguished     by     the     fact    that  pulmonary     abscess 

they  are  Gram-decolorizing.  (X  about  1000).  (Boston.) 

(5)  Bacillus  of  Influenza. — This  is  the  etiologic  factor 
in  true  influenza,  although  conditions  which  are  clinic- 
ally similar  or  identical  may  be  caused  by  the  pneu- 


FiG.  25. — Bacillus  of  influenza;  cover-glass  preparation  of  sputum 
from  a  case  of  influenza,  showing  the  bacilli  in  leukocytes;  highly 
magnified  (Pfeifler). 

mococcus,    streptococcus,    or   Micrococcus  catarrhalis. 
It  is  present,  often  in  large  numbers,  in  the  nasal  and 


90  THE    SPUTUM 

bronchial  secretions,  and  is  also  found  in  the  local  lesions 
following  influenza.  Chronic  infection  by  influenza 
bacilli  may  be  mistaken  clinically  for  tuberculosis,  and 
they  should  be  searched  for  in  all  cases  of  obstinate 
chronic  bronchitis. 

Their  recognition  depends  upon  the  facts  that  they 
are  extremely  small  bacilli;  that  most  of  them  lie  within 
the  pus-cells;  that  their  ends  stain  more  deeply  than  their 
centers,  sometimes,  giving  the  appearance  of  minute 
diplococci;  and  that  they  are  decolorized  by  Gram's 
method  of  staining  (Figs.  25  and  212). 

They  are  well  stained  by  dilute  fuchsin  or  by  Pappen- 
heim's  pyronin-methyl  green,  but  are  more  certainly 
recognized  by  Gram's  method  with  the  pyronin-methyl 
green  for  counterstain. 

(6)  Bacillus  pertussis. — The  whooping-cough  bacillus 
is  a  minute,  ovoid,  Gram-negative  bacillus  which  stains 
feebly  with  the  ordinary  dyes,  and  sometimes,  though 
not  usually,  lies  within  pus  cells.  It  can  be  demonstrated 
by  the  method  given  for  the  influenza  bacillus. 

(7)  Micrococcus  catarrhalis. — This  organism  is  fre- 
quently present  in  the  sputum  in  inflammatory  condi- 
tions of  the  respiratory  tract  resembling  influenza. 
It  is  sometimes  present  in  the  nasal  secretions  in  health. 
It  is  a  Gram-negative  diplococcus,  frequently  intra- 
cellular, and  can  be  distinguished  from  the  meningo- 
coccus and  gonococcus  only  by  means  of  cultures  (Fig. 
26).  The  staining  method  recommended  for  the  influ- 
enza bacillus  is  best.  It  grows  readily  on  ordinary 
mQdia. 

2.  Cells. — These  include  pus-corpuscles,  epithelial 
cells,  and  red  blood-corpuscles. 


MICROSCOPIC   EXAMINATION  9I 

(i)  Pus-corpuscles  are  present  in  every  sputum,  and 
at  times  the  sputum  may  consist  of  little  else.  They  are 
the  polymorphonuclear  leukocytes  of  the  blood,  and 
appear  as  rounded  cells  with  several  nuclei  or  one  very 
irregular  nucleus  (Plate  II,  Fig.  3).  They  are  often 
much-  disintegrated.     Occasional  lymphocytes  are  usu- 


Fig.   26.-  — Micrococcus  catarrhalis  in  smear  from  spvitum  (F.  T.  Lord; 
photo  by  L.  S.  Brown). 

ally    present.     Their    predominance   is    suggestive   of 
early  or  mild  tuberculosis. 

Eosinophilic  cells  are  quite  constantly  found  in  large 
numbers  in  the  sputum  of  bronchial  asthma  near  the 
time  of  the  paroxysm,  and  constitute  one  of  the  most 
distinctive  features  of  the  sputum  of  this  disease.  They 
resemble    ordinary   pus-corpuscles,    except    that    their 


92  THE    SPUTUM 

cytoplasm  is  filled  with  coarse  granules  having  a  marked 
affinity  for  eosin.  It  is  worthy  of  note  that  many  of 
them,  sometimes  the  majority,  are  mononuclear. 
Large  numbers  of  free  granules,  derived  from  dis- 
integrated cells,  are  also  found  (Plate  II,  Fig.  2). 

Ordinary  pus-cells  are  easily  recognized  in  sputum 
stained  by  any  of  the  methods  already  given.  For 
eosinophilic  cells,  some  method  which  includes  eosin 
must  be  used.  A  simple  method  is  to  stain  the  dried 
and  fijced  film  two  or  three  minutes  with  saturated 
solution  of  eosin,  and  then  one-half  to  one  minute  with 
LofHer's  methylene  blue;  nuclei  and  bacteria  will  be  blue, 
eosinophilic  granules  bright  red.  Either  Wright's  or 
Jenner's  stain  (p.  313)  will  be  found  satisfactory. 

(2)  Epithelial  cells  may  come  from  any  part  of  the 
respiratory  tract.  A  few  are  always  present,  since  des- 
quamation of  cells  goes  on  constantly.  Their  recogni- 
tion is  important  chiefly  as  an  aid  in  deciding  upon  the 
source  of  the  portion  of  the  sputum  in  which  they  are 
found.  In  suspected  lung  conditions  it  is  manifestly 
useless  to  study  material  from  the  nose  only,  yet  this 
is  not  infrequently  done.  They  have  little  diagnostic 
value,  although  a  considerable  excess  would  indicate  a 
pathologic  condition  at  the  site  of  their  origin.  Any 
of  the  stains  mentioned  above  will  show  them,  and  they 
can  usually  be  identified  in  unstained  sputum.  In 
general,  three  forms  are  found : 

(a)  Squamous  Cells. — Large,  flat,  polygonal  cells  with 
a  comparatively  small  nucleus  (Fig.  27,  i).  They  come 
from  the  upper  air-passages,  and  are  especially  numerous 
in  laryngitis  and  pharyngitis.  They  are  frequently 
studded  with  bacteria — most  commonly  diplococci. 


MICROSCOPIC  EXAMINATION 


93 


(b)  Cylindric  Cells  from  the  Nose,  Trachea,  and  Bronchi 
(Fig.  27,  /,  //). — These  are  not  usually  abundant,  and, 
as  a  rule,  they  are  not  identified  because  much  altered 
from  their  original  form,  being  usually  round  and 
swollen.  Whan  very  fresh,  they  may  retain  their 
cylindric  form,  sometimes  bearing  cilia  in  active 
motion. 


Fig.  27. — Different  morphologic  elements  of  the  sputum  (un- 
stained): a,  b,  c.  Pulmonary  or  alveolar  epithelium — a,  with  normal 
lung  pigment  (carbon);  6,  with  fat-droplets;  c,  with  myelin  globules; 
d,  pus-corpuscles;  e,  red  blood-corpuscles;  /,  cylindric  beaker-shaped 
bronchial  epithelial  cells;  g,  free  myelin  globules;  h,  ciliated  epithelium 
of  different  kinds  from  the  nose,  altered  by  coryza;  i,  squamous  cells 
from  the  pharynx  (after  Bizzozero). 

(c)  Alveolar  Cells. — Rather  large,  round,  or  oval  cells, 
three  to  six  times  the  diameter  of  a  red  corpuscle, 
with  one  or  two  round  nuclei  (Fig.  27).  Their  source  is 
presumably  the  pulmonary  alveoli.  It  is  probable  that 
many  of  the  cells  which  have  been  included  in  this 
group  are  really  large  mononuclear  leukocytes. 


94  THE    SPUTUM 

(3)  Red  blood-corpuscles  may  be  present  in  small 
numbers  in  almost  any  sputum.  When  fairly  constantly 
present  in  considerable  numbers,  they  are  suggestive  of 
phthisis.  The  corpuscles,  when  fresh,  can  easily  be 
recognized  in  unstained  sputum  or  may  be  shown  by 
any  of  the  staining  methods  which  include  eosin. 
They  are,  however,  commonly  so  much  degenerated 
as  to  be  unrecognizable  and  often  only  altered  blood- 
pigment  is  left.  Ordinarily,  blood  in  the  sputum  is 
sufl&ciently  recognized  with  the  naked  eye. 

IIL  CHEMIC  EXAMINATION 

There  is  little  to  be  learned  from  a  chemic  examina- 
tion, and  it  is  rarely  undertaken.  Recently,  however,  it 
has  been  shown  that  the  presence  or  absence  of  albumin 
may  have  clinical  significance.  x\lbumin  is  almost 
constantly  present  in  the  sputum  in  pneumonia,  pul- 
monary edema,  and  tuberculosis.  It  is  usually  absent 
in  bronchitis.  A  test  for  albumin  may,  therefore,  be 
of  some  value  in  distinguishing  between  bronchitis  and 
tuberculosis.     It  is  carried  out  as  follows : 

Method  for  Albumin  in  Sputum. — i.  To  10  c.c.  of  the 
sputum  add  30  c.c.  of  i  per  cent,  acetic  acid  and  shake 
until  thoroughly  mixed.  This  may  be  done  in  a  stop- 
pered bottle.  Dilution  and  addition  of  acetic  acid  pre- 
cipitates the  mucus. 

2.  Filter  through  filter-paper. 

3.  Test  the  filtrate  for  albumin  qualitatively  and  quan- 
titatively, as  described  in  the  chapter  upon  the  urine. 

Active  cases  of  phthisis,  whether  early  or  far  ad- 
vanced, generally  show  0.2  per  cent,  or  more  albumin; 
slightly   active   cases,   less   than  0.2   per   cent.     The 


THE    SPUTUM   IN   DISEASE  95 

sputum  must  be  fresh,  otherwise  a  negative  reaction 
may  have  changed  to  positive  owing  to  disintegration 
of  cells. 

IV.  THE  SPUTUM  IN  DISEASE 

Strictly  speaking,  any  appreciable  amount  of  sputum 
is  abnormal.  A  great  many  healthy  persons,  however, 
raise  a  small  quantity  each  morning,  owing  chiefly  to 
the  irritation  of  inhaled  dust  and  smoke.  Although 
not  normal,  this  can  hardly  be  spoken  of  as  pathologic. 
It  is  particularly  frequent  in  city  dwellers  and  in  those 
who  smoke  cigarettes  to  excess.  In  the  latter  the 
amount  is  sometimes  so  great  as  to  arouse  suspicion  of 
tuberculosis.  Such  ''normal  morning  sputum"  gen- 
erally consists  of  small,  rather  dense,  mucoid  masses, 
translucent-white,  or,  when  due  to  inhaled  smoke,  gray 
in  color.  Microscopically,  there  are  a  few  pus-cor- 
puscles, and,  usually,  many  alveolar  cells,  both  of  which 
may  contain  carbon  particles.  The  alveolar  cells  com- 
monly show  myelin  degeneration,  and  free  myelin  glob- 
ules may  be  present  in  large  numbers.  Saprophytic 
bacteria  may  be  present,  but  are  not  abundant. 

1.  Acute  Bronchitis. — There  is  at  first  a  small 
amount  of  tenacious,  almost  purely  mucoid  sputum, 
frequently  blood  streaked.  This  gradually  becomes 
more  abundant,  mucopurulent  in  character,  and  yellow- 
ish or  gray  in  color.  At  first  the  microscope  shows  a 
few  leukocytes  and  alveolar  and  bronchial  cells;  later 
the  leukocytes  become  more  numerous.  Bacteria  are 
not  usually  abundant. 

2.  Chronic  Bronchitis. — The  sputum  is  usually 
abundant,  mucopurulent,  and  yellowish  or  yellowish- 


96  THE   SPUTUM 

green  in  color.  Nummular  masses  like  those  of  tuber- 
culosis are  sometimes-  seen.  Microscopically,  there 
are  great  numbers  of  leukocytes,  often  much  disinte- 
grated. Epithelium  is  not  abundant.  Bacteria  of 
various  kinds,  especially  staphylococci,  are  usually 
numerous. 

In  fibrinous  bronchitis  there  are  found,  in  addition, 
fibrinous  casts,  usually  of  medium  size. 

In  the  chronic  bronchitis  accompanying  long-con- 
tinued passive  congestion  of  the  lungs,  as  in  poorly 
compensated  heart  disease,  the  sputum  may  assume  a 
rusty  brown  color,  owing  to  presence  of  large  numbers 
of  the  "heart-failure  cells"  previously  mentioned. 

3.  Bronchiectasis. — When  there  is  a  single  large 
cavity,  the  sputum  is  very  abundant  at  intervals — 
sometimes  as  high  as  a  liter  in  twenty-four  hours — and 
has  a  very  offensive  odor.  It  is  thinner  than  that  of 
chronic  bronchitis,  and  upon  standing  separates  into 
three  layers  of  pus,  mucus,  and  frothy  serum.  It  con- 
tains great  numbers  of  miscellaneous  bacteria. 

4.  Gangrene  of  the  Lung. — The  sputum  is  abun- 
dant, fluid,  very  offensive,  and  brownish  in  color.  It 
separates  into  three  layers  upon  standing — a  brown 
deposit,  a  clear  fluid,  and  a  frothy  layer.  Microscopic- 
ally, few  cells  of  any  kind  are  found.  Bacteria  are  ex- 
tremely numerous;  among  them  may  sometimes  be 
found  an  acid-fast  bacillus  probably  identical  with  the 
smegma  bacillus.  As  before  stated,  elastic  fibers  are 
usually  present  in  large  fragments. 

5.  Pulmonary  Edema. — Here  there  Is  an  abundant, 
watery,  frothy  sputum,  varying  from  faintly  yellow  or 
pink  to  dark  brown  in  color;  a  few  leukocytes  and 


THE    SPUTUM   IN   DISEASE  97 

epithelial  cells  and  varying  numbers  of  red  blood-cor- 
puscles are  found  with  the  microscope. 

6.  Bronchial  Asthma. — The  sputum  during  and 
following  an  attack  is  scanty  and  very  tenacious.  Most 
characteristic  is  the  presence  of  Curschmann's  spirals, 
Charcot-Leyden  crystals,  and  eosinophilic  leukocytes. 

7.  Croupous  Pneumonia. — Characteristic  of  this 
disease  is  a  scanty,  rusty  red,  very  tenacious  sputum, 
containing  red  corpuscles  or  altered  blood-pigment,  leu- 
kocytes, epithelial  cells,  usually  many  pneumococci,  and 
often  very  small  fibrinous  casts.  This  sputum  is  seen 
during  the  stage  of  red  hepatization.  During  resolu- 
tion the  sputum  assumes  the  appearance  of  that  of 
chronic  bronchitis.  When  pneumonia  occurs  during  the 
course  of  a  chronic  bronchitis,  the  characteristic  rusty 
red  sputum  may  not  appear. 

8.  Pulmonary  Tuberculosis. — The  sputum  is  vari- 
able. In  the  earliest  stages  it  may  appear  only  in  the 
morning,  and  is  then  scanty  and  almost  purely  mucoid, 
with  an  occasional  yellow  flake;  or  there  may  be  only 
one  very  small  mucopurulent  mass.  When  the  quan- 
tity is  small,  there  may  be  no  cough,  the  sputum  reach- 
ing the  larynx  by  action  of  the  bronchial  cilia.  This  is 
not  well  enough  recognized  by  practitioners.  A  care- 
ful inspection  of  all  the  sputum  brought  up  by  the  pa- 
tient on  several  successive  days,  and  a  microscopic 
examination  of  all  yellow  portions,  will  not  infrequently 
establish  a  diagnosis  of  tuberculosis  when  physical  signs 
are  negative."  Intelligent  cooperation  of  the  patient  is 
essential  in  such  cases.  Tubercle  bacilli  will  sometimes 
be  found  in  large  numbers  at  this  stage.  Blood- 
streaked  sputum  is  strongly  suggestive  of  tuberculosis, 


98  THE    SPUTUM 

and  is  more  common  in  the  early  stages  than  later.     It 
usually  indicates  an  advancing  process. 

The  sputum  of  more  advanced  cases  resembles  that 
of  chronic  bronchitis,  with  the  addition  of  tubercle  ba- 
cilli and  elastic  fibers.  Nummular  masses — circular, 
"coin-like"  disks,  which  sink  in  water — may  be  seen. 
Caseous  particles  containing  immense  numbers  of  the 
bacilli  are  common.  Far-advanced  cases  with  old 
cavities  often  show  rather  firm,  spheric  or  ovoid  gray- 
ish masses  in  a  thin  fluid — the  so-called  "globular  spu- 
tum." These  globular  masses  usually  contain  many 
tubercle  bacilli.  Considerable  hemorrhages  are  not  in- 
frequent, and  for  some  time  thereafter  the  sputum  may 
contain  clots  of  blood  or  be  colored  brown. 


CHAPTER  II 
THE  URINE 

Preliminary  Considerations. — The  urine  is  an  ex- 
tremely complex  aqueous  solution  of  various  organic 
and  inorganic  substances.  Most  of  the  substances  are 
either  waste-products  from  the  body  metabolism  or 
products  derived  directly  from  the  foods  eaten.  Nor- 
mally, the  total  amount  of  solid  constituents  carried  off 
in  twenty-four  hours  is  about  60  Gm.,  of  which  the  or- 
ganic substances  make  up  about  35  Gm.  and  the  in- 
organic about  25  Gm. 

The  most  important  organic  constituents  are  urea, 
uric  acid,  creatinin  and  ammonia.  Urea  constitutes 
about  one-half  of  all  the  solids,  or  about  30  Gm.  in 
twenty-four  hours. 

The  chief  inorganic  constituents  are  the  chlorids, 
phosphates,  and  sulphates.  The  chlorids,  practically 
all  in  the  form  of  sodium  chlorid,  make  up  about  one- 
half  of  the  inorganic  substances,  or  about  13  Gm.,  in 
twenty-four  hours. 

Certain  substances  appear  in  the  urine  only  in  patho- 
logic conditions.  The  most  important  of  these  are  pro- 
teins, sugars,  acetone,  and  related  substances,  bile, 
hemoglobin,  and  the  diazo  substances. 

In  addition  to  the  substances  in  solution  all  urines 
contain  various  microscopic  structures. 

While,  under  ordinary  conditions,  the  composition  of 

99 


lOO  THE    URINE 

urine  does  not  vary  much  from  day  to  day,  it  varies 
greatly  at  different  hours  of  the  same  day.  It  is  evi- 
dent, therefore,  that  the  collection  of  the  specimen  is 
important  and  that  no  quantitative  test  can  he  of  value  un- 
less a  sample  of  the  mixed  twenty-four-hour  urine  he  used. 
The  patient  should  be  instructed  to  void  all  the  urine 
during  the  twenty-four  hours  into  a  clean  vessel  kept  in  a 
cool  place,  to  mix  it  well,  to  measure  the  whole  quantity, 
and  to  bring  8  or  more  ounces  for  examination.  In 
order  to  avoid  annoying  misunderstandings,  it  is  well 
to  make  these  directions  specific,  telling  him  to  empty 
the  bladder  at  a  specified  time,  say  8  a.m.  and  to  discard 
this  urine,  to  save  all  the  urine  voided  up  to  8  a.m.  of 
the  next  day  and  at  that  time  to  empty  the  bladder 
whether  he  feels  the  need  for  it  or  not,  and  to  add  this 
final  amount  to  the  quantity  collected.  WTien  it  is  de- 
sired to  make  only  qualitative  tests,  as  for  albumin  or 
sugar,  a  "sample"  voided  at  random  will  answer.  It 
should  be  remembered,  however,  that  urine  passed 
about  three  hours  after  a  meal  is  most  likely  to  contain 
pathologic  substances.  That  voided  first  in  the  morn- 
ing is  least  likely  to  contain  them.  To  diagnose  cyclic 
albuminuria  samples  obtained  at  various  periods  during 
the  twenty-four  hours  must  be  examined. 

The  urine  must  be  examined  while  fresh.  Decom- 
position sets  in  rapidly,  especially  in  warm  weather,  and 
greatly  interferes  with  all  the  examinations.  Decom- 
position may  be  delayed  by  adding  5  gr.  of  boric  acid 
(as  much  of  the  powder  as  can  be  heaped  upon  a  ten- 
cent  piece)  for  each  4  ounces  of  urine.  Formalin,  in 
proportion  of  i  drop  to  4  ounces,  is  also  an  efiicient  pre- 
servative, but  if  larger  amounts  be  used,  it  may  give 


THE    URINE  lOI 

reactions  for  sugar  and  albumin,  and  is  likely  to  cause  a 
precipitate  which  greatly  interferes  with  the  micro- 
scopic examination.  Thymol,  toluol,  and  chloroform 
are  likewise  much  used.  The  use  of  thymol  is  very 
convenient.  A  small  lump,  floating  upon  the  surface, 
wall  preserve  a  bottle  of  urine  for  some  days,  but  enough 
may  dissolve  to  simulate  the  albumin  reaction.  The 
chief  objection  to  toluol  is  the  fact  that  it  floats  upon  the 
surface,  and  the  urine  must  be  pipeted  from  beneath  it. 
Chloroform  is  probably  the  least  satisfactory.  It  re- 
duces Fehling's  solution ;  and  it  settles  to  the  bottom  in 
the  form  of  globules  which  it  is  impossible  to  avoid  when 
removing  the  sediment  for  microscopic  examination. 
One  of  these  preservatives  may  be  placed  in  the  vessel 
when  collection  of  the  twenty-four-hour  sample  is  begun. 
Whenever  possible  the  urine  should  be  kept  on  ice. 

Normal  and  abnormal  pigments,  which  interfere  with 
certain  of  the  tests,  can  be  removed  by  filtering  the  urine 
through  animal  charcoal,  or  precipitating  with  a  solu- 
tion of  normal  acetate  of  lead  (sugar  of  lead)  or  with 
powdered  lead  acetate  in  substance  and  filtering. 

Certain  cloudy  urines  cannot  be  clarified  by  ordinary 
filtration  through  paper,  particularly  when  the  cloudi- 
ness is  due  to  bacteria.  Such  urines  can  usually  be 
rendered  perfectly  clear  by  adding  a  small  amount  of 
purified  talc  or  infusorial  earth,  shaking  well,  and 
filtering. 

A  suspected  fluid  can  be  identified  as  urine  by  detect- 
ing any  considerable  quantity  of  urea  in  it  (see  p.  136). 
Traces  of  urea  may,  however,  be  met  with  in  ovarian 
cyst  fluid,  while  urine  from  very  old  cases  of  hydro- 
nephrosis may  contain  little  or  none. 


I02  THE    URINE 

The  frequency  of  micturition  is  often  suggestive  in 
diagnosis.  Whether  it  is  unduly  frequent  can  best  be 
ascertained  by  asking  the  patient  whether  he  has  to  get 
up  at  night  to  urinate.  Increased  frequency  may  be 
due  to  restlessness;  to  increased  quantity  of  urine;  to 
irritability  of  the  bladder,  usually  an  evidence  of  cys- 
titis; to  obstruction  ("retention  with  overflow");  or  to 
paralysis  of  the  sphincter. 

Clinical  examination  of  the  urine  may  conveniently  be 
considered  under  five  heads:  I.  General  characteristics. 
II.  Functional  tests.  III.  Chemic  examination.  IV. 
Microscopic  examination.     V.  The  urine  in  disease. 

I.   GENERAL  CHARACTERISTICS 

1.  Quantity. — The  quantity  passed  in  twenty-four 
hours  varies  greatly  with  the  amount  of  liquids  ingested, 
perspiration,  etc.  The  normal  may  be  taken  as  looo  to 
1500  c.c,  or  35  to  50  ounces  for  an  adult  in  this  country. 
German  writers  give  higher  figures.  For  children  the 
amount  is  somewhat  greater  in  proportion  to  body 
weight. 

The  quantity  is  increased  (polyuria)  during  absorp- 
tion of  large  serous  effusions  and  in  many  nervous  con- 
ditions. It  is  usually  much  increased  in  chronic  inter- 
stitial nephritis,  diabetes  insipidus,  and  diabetes  melli- 
tus.  In  these  conditions  a  permanent  increase  in 
amount  of  urine  is  fairly  constant — a  fact  of  much 
value  in  diagnosis.  In  diabetes  mellitus  the  urine  may, 
though  rarely,  reach  the  enormous  amount  of  50  liters. 

The  quantity  is  decreased  (oliguria)  in  severe  diarrhea ; 
in  fevers;  in  all  conditions  which  interfere  with  circula- 
tion in  the  kidney,  as  poorly  compensated  heart  disease ; 


GENERAL    CHARACTERISTICS  IO3 

in  the  parenchymatous  forms  of  nephritis;  and  during 
accumulation  of  fluid  in  the  serous  cavities.  In  uremia 
the  urine  is  usually  very  greatly  decreased  and  may  be 
entirely  suppressed  (anuria). 

Ordinarily,  more  urine  is  voided  during  the  day  than 
during  the  night,  the  normal  ratio  being  about  loo  to  50 
or  60.  In  certain  diseases,  notably  arteriosclerosis  and 
cardiac  and  renal  disease,  conditions  are  reversed,  and 
the  night  urine  (7  p.m.  to  7  a.m.)  equals  or  exceeds  that 
passed  during  the  day. 

2.  Color. — This  varies  considerably  in  health,  and 
depends  largely  upon  the  quantity  of  urine  voided,  di- 
lute urines  being  pale  and  concentrated  urines,  highly 
colored.  The  usual  color  is  yellow  or  reddish  yellow, 
due  to  the  presence  of  several  pigments,  chiefly  uro- 
chrome,  which  is  yellow.  Traces  of  hematoporphyrin, 
uroerythrin,  and  urobilin  are  frequent.  Uroerythrin 
is  chiefly  responsible  for  the  deep  reddish  tinge  of  urine 
in  acute  fevers.  Urobilin  and  hematoporphyrin  have 
clinical  significance  and  are  discussed  later  (see  p.  184). 
Acid  urine  is  generally  darker  than  alkaline.  For 
the  sake  of  uniformity  in  recording  the  color,  Vogel's 
scale  is  widely  used,  the  urine  being  filtered  and  ex- 
amined by  transmitted  light  in  a  glass  3  or  4  inches  in 
diameter.  This  scale  uses  nine  colors:  pale  yellow, 
light  yellow,  yellow,  reddish  yellow,  yellowish  red,  red, 
brownish  red,  reddish  brown,  and  brownish  black. 

Color  is  sometimes  greatly  changed  by  abnormal 
pigments.  Blood-pigment  gives  a  red  or  brown,  smoky 
color.  Urine  containing  bile  is  yellowish  or  brown,  with 
a  yellow  foam  when  shaken.  It  may  assume  a  greenish 
hue  after  standing,  owing  to  oxidation  of  bilirubin  into 


I04  THE    URINE 

biliverdin.  Ingestion  of  small  amounts  of  methylene 
blue  gives  a  pale  green;  large  amounts  give  a  marked 
greenish  blue.  Santonin  produces  a  yellow;  rhubarb, 
senna,  cascara,  and  some  other  cathartics,  a  brown 
color;  these  change  to  red  upon  addition  of  an  alkali, 
and  if  the  urine  be  alkaline  when  voided,  may  cause 
suspicion  of  hematuria.  A  bright  pink  or  red  color 
appearing  when  the  urine  is  alkalinized  may  be  due  to 
phenolphthalein.  Thymol  gives  a  yellowish  green. 
Following  poisoning  from  phenol  and  related  drugs  the 
urine  may  have  a  normal  color  when  voided,  but  be- 
comes olive  green  to  brownish  black  upon  standing.  In 
susceptible  individuals  therapeutic  doses  of  creosote,  or 
absorption  from  carbolized  dressings,  may  cause  this 
change.  Urine  which  contains  melanin,  as  sometimes 
in  melanotic  tumors,  and  very  rarely  in  wasting  dis- 
eases, also  becomes  brown  or  black  upon  long  stand- 
ing. A  similar  darkening  upon  exposure  to  the  air 
occurs  in  alkaptonuria  (see  p.  183).  A  milky  color  may 
be  due  to  presence  of  chyle,  or  milk  may  have  been 
added  by  a  malingering  patient. 

A  pale  greenish  urine  with  high  specific  gravity 
strongly  suggests  diabetes  mellitus. 

3.  Transparency. — Freshly  passed  normal  urine  is 
clear.  Upon  standing,  a  faint  cloud  of  mucus,  leuko- 
cytes, and  epithelial  cells  settles  to  the  bottom — the  so- 
called  "nubecula."  This  is  more  abundant  in  women 
owing  to  vaginal  cells  and  mucus.  In  urines  of  high 
specific  gravity  it  may  float  near  the  middle  of  the  fluid. 

Abnormal  cloudiness  is  usually  due  to  presence  of 
phosphates,  urates,  pus,  blood,  or  bacteria.  Epithelial 
cells   and   tube-casts   are   rarely  present  in   sufficient 


GENERAL    CHARACTERISTICS  105 

number  to  produce  more  than  a  slight  cloudiness 
although  they  may  add  to  turbidity  due  to  other 
causes. 

Amorphous  phosphates  are  precipitated  in  neutral 
or  alkaline  urine.  They  form  a  white  cloud  and  sedi- 
ment, which  disappear  upon  addition  of  an  acid. 

Amorphous  urates  are  precipitated  only  in  acid  urine. 
They  form  a  white  or  pink  cloud  and  sediment  ("brick- 
dust  deposit"),  which  disappear  upon  heating. 

Pus  resembles  amorphous  phosphates  to  the  naked 
eye.  Its  nature  is  easily  recognized  with  the  micro- 
scope, or  by  adding  a  strong  solution  of  caustic  soda  to 
the  sediment,  which  is  thereby  transformed  into  a  gela- 
tinous mass  (Donne's  test). 

Blood  gives  a  reddish  or  brown,  smoky  color,  and 
may  be  recognized  with  the  microscope  or  by  tests  for 
hemoglobin. 

Bacteria,  when  present  in  great  numbers,  give  a  uni- 
form cloud,  which  cannot  be  removed  by  ordinary  filtra- 
tion.    They  are  detected  with  the  microscope. 

The  cloudiness  of  decomposing  urine  Is  due  mainly 
to  precipitation  of  phosphates  and  multiplication  of 
bacteria. 

4.  Odor. — The  characteristic  aromatic  odor  has 
generally  been  attributed  to  volatile  acids,  but  a  newly 
discovered  substance,  called  "urinod,"  has  more  re- 
cently been  held  responsible.  The  odor  is  most  marked 
in  concentrated  urines.  During  decomposition  the 
odor  becomes  ammoniacal.  A  fruity  odor  is  sometines 
noted  in  diabetes,  due  probably  to  acetone.  Urine 
which  contains  cystin  may  develop  an  odor  of  sul- 
phureted  hydrogen  during  decomposition. 


I06  THE    URINE 

Various  articles  of  diet  and  drugs  impart  peculiar 
odors.  Notable  among  these  are  asparagus,  which 
gives  a  characteristic  offensive  odor,  and  turpentine, 
which  imparts  an  odor  somewhat  suggesting  that  of 
violets. 

5.  Reaction. — Normally,  the  mixed  twenty-four- 
hour  urine  is  slightly  acid  in  reaction.  The  acidity 
sometimes  increases  for  a  time  after  the  urine  is  voided, 
the  so-called  "acid  fermentation."  The  acidity  was 
formerly  held  to  be  due  wholly  to  acid  phosphates,  but 
Folin  has  shown  that  the  acidity  of  a  clear  urine  is 
ordinarily  greater  than  the  acidity  of  all  the  phosphates, 
the  excess  being  due  to  free  organic  acids.  Individual 
samples  may  be  slightly  alkaline,  especially  after  a  full 
meal;  or  they  may  be  amphoteric,  turning  red  litmus- 
paper  blue  and  blue  paper  red,  owing  to  presence  of  both 
alkaline  and  acid  phosphates.  The  reaction  is  ordi- 
narily determined  by  means  of  litmus-paper,  which, 
however,  is  worthless  unless  of  good  quality.  That  put 
up  in  vials  by  Squibb  can  be  recommended. 

Acidity  is  increased  after  administration  of  certain 
drugs,  by  excess  of  protein  in  the  diet,  and  whenever  the 
urine  is  concentrated  from  any  cause,  as  in  fevers.  A 
strongly  acid  urine  may  cause  frequent  micturition 
because  of  its  irritation.  This  is  often  an  important 
factor  in  the  troublesome  enuresis  of  children. 

The  urine  always  becomes  alkaline  upon  long  stand- 
ing, owing  to  decomposition  of  urea  with  formation  of 
ammonia.  If  markedly  alkaline  when  voided,  it  usu- 
ally indicates  such  "ammoniacal  decomposition"  in  the 
bladder,  which  is  the  rule  in  chronic  cystitis,  especially 
that  due  to  paralysis  or  obstruction.     Alkalinity  due  to 


GENERAL   CHARACTERISTICS  107 

ammonia,  volatile  alkalinity,  can  be  recognized  by  the 
odor  or  by  the  fact  that  Htmus-paper  turned  blue  by  the 
urine  again  becomes  red  upon  gentle  heating,  or  that  the 
paper  will  turn  blue  when  held  in  the  steam  over  the 
boiling  urine.  A  second  form  of  alkalinity,  fixed  alka- 
linity, is  due  to  alkaline  salts,  and  is  often  observed  dur- 
ing frequent  vomiting,  after  the  crisis  of  pneumonia,  in 
various  forms  of  anemia,  during  digestion  of  full  meals, 
after  abundant  eating  of  fruits,  and  after  administration 
of  certain  drugs,  especially  salts  of  vegetable  acids. 

Quantitative  estimation  of  acidity  of  urine  is  not  of 
much  clinical  value.  WTien,  however,  it  is  desired  to 
make  it,  the  method  of  Folin  will  be  found  satisfactory. 
In  every  case  the  sample  must  be  from  the  mixed 
twenty-four-hour  urine  and  as  fresh  as  possible. 

Folin's  Method. — Into  a  small  flask  measure  25  c.c.  of 
the  urine  and  add  i  or  2  drops  0.5  per  cent,  alcoholic  solu- 
tion of  phenolphthalein  and  15  or  20  Gm.  of  neutral  potas- 
sium oxalate.  Shake  for  a  minute,  and  immediately 
titrate  with  decinormal  sodium  hydroxid,  shaking  mean- 
while, until  the  first  permanent  pink  appears.  Read  off 
from  the  buret  the  amount  of  decinormal  sodium  hydroxid 
solution  added,  and  calculate  the  number  of  cubic  centi- 
meters which  would  be  required  for  the  entire  twenty-four 
hours'  urine.  Most  estimations  run  between  25  and  40  c.c. 
of  decinormal  solution  for  100  c.c.  of  urine.  Folin  places 
the  normal  acidity  for  the  twenty-four  hour  specimen  at 
554  to  669  c.c.  of  decinormal  solution,  but  most  other 
authors  give  lower  figures.     Much  depends  upon  the  diet. 

6.  Specific  Gravity. — In  a  general  way  this  varies 
inversely  with  the  quantity  of  urine.  The  normal  aver- 
age is  about  1. 01 7  to  1.020.     Samples  of  urine  taken  at 


io8 


THE   URINE 


random  may  go  far  above  or  below  these  figures,  hence 
a  sample  of  the  mixed  twenty-four-hour  urine  should 
always  be  used. 

Pathologically,  it  may  vary  from  i.ooi  to  1.060.  It  is 
low  in  chronic  interstitial  nephritis,  diabetes  insipidus, 
and  many  functional  nervous  disorders.  It  is  high  in 
fevers  and  in  parenchymatous  disease  of  the  kidney.  In 
any  form  of  nephritis  a  sudden  fall  without  a  corre- 
sponding increase  in  quantity  of  urine  may  foretell  ap- 

(9k 


Fig.  28. — Squibb's  urinometer  with  thermometer  and  cylinder. 

proaching  uremia.  It  is  highest  in  diabetes  mellitus. 
A  high  specific  gravity  when  the  urine  is  not  highly  col- 
ored, or  when  the  quantity  is  above  the  normal,  should 
lead  one  to  suspect  this  disease.  A  normal  specific 
gravity  does  not,  however,  exclude  it. 

The  specific  gravity  is  most  conveniently  estimated 
by  means  of  the  urinometer  (Fig.  28).  Squibb's  urin- 
ometer is  adjusted  to  give  accurate  readings  at  22.5° C; 
most  other  instruments,  at  i5°C.  If  the  urine  be 
brought  to  about  the  right  temperature,  a  correction  for 


GENERAL   CHARACTERISTICS 


109 


temperature  will  seldom  be  necessary  in  clinical  work. 
For  accuracy,  however,  it  is  necessary  to  add  0.00 1  to 
the  urinometer  reading  for  each  3°C.  above  the  tem- 
perature for  which  the  urinometer  is  standardized,  and 
to  subtract  o.ooi  for  each  3°C.  below  that  point.  Care 
should  be  taken  that  the  urinometer  does  not  touch  the 
side  of  the  tube,  and  that  air-bubbles  are  removed  from 


Fig.  29. — Saxe's  urinopyknometer  and  jar  for  same. 


the  surface  of  the  urine.  Bubbles  are  easily  removed 
with  a  strip  of  filter-paper.  With  most  instruments  the 
reading  is  taken  from  the  bottom  of  the  meniscus.  A 
long  scale  on  the  stem  is  desirable,  because  of  the  greater 
ease  of  accurate  reading.  Many  of  the  urinometers  on 
the  market  are  too  small  to  be  of  any  real  value. 


no  THE    URINE 

One  frequently  wishes  to  ascertain  the  specific  gravity 
of  quantities  of  fluid  too  small  to  float  a  urinometer. 
A  simple  device  for  this  purpose,  which  requires  only 
about  3  c.c.  and  is  very  satisfactory  in  clinical  work,  has 
been  designed  by  Saxe  (Fig.  29).  The  urine  is  placed  in 
the  bulb  at  the  bottom,  the  instrument  is  floated  in  dis- 
tilled water,  and  the  specific  gravity  is  read  off  from  the 
scale  upon  the  stem. 

7.  Total  SoIids.^An  estimation  of  the  total 
amount  of  solids  which  pass  through  the  kidneys  in 
twenty-four  hours  is,  in  practice,  one  of  the  most  use- 
ful of  urinary  examinations.  The  normal  for  a  man 
of  150  pounds  is  about  60  Gm.,  or  950  gr.  The  prin- 
cipal factors  which  influence  this  amount  are  body 
weight  (except  with  excessive  fat),  diet,  exercise,  and 
age,  and  these  should  be  considered  in  making  an 
estimation.  After  about  the  forty-fifth  year  it  be- 
comes gradually  less ;  after  the  seventy-fifth  it  is  about 
one-half  the  amount  given. 

In  disease  the  amount  of  solids  depends  mainly  upon 
the  activity  of  metabolism  and  the  ability  of  the  kidneys 
to  excrete.  An  estimation  of  the  solids,  therefore, 
furnishes  an  important  clue  to  the  functional  efficiency 
of  the  kidneys.  The  kidneys  bear  much  the  same 
relation  to  the  organism  as  does  the  heart;  they  cause 
no  direct  harm  so  long  as  they  are  capable  of  perform- 
ing the  work  required  of  them.  When,  however, 
through  either  organic  disease  or  functional  inactivity, 
they  fail  to  carry  off  their  proportion  of  the  waste- 
products  of  the  body,  some  of  these  products  must  either 
be  eliminated  through  other  organs,  where  they  cause 
irritation  and  disease,  or  be  retained  within  the  body. 


GENERAL    CHARACTERISTICS  III 

where  they  act  as  poisons.  The  great  importance  of 
these  poisons  in  production  of  distressing  symptoms 
and  even  organic  disease  is  not  well  enough  recognized 
by  most  pralJitioners.  Disappearance  of  unpleasant 
and  perplexing  symptoms  as  the  urinary  solids  rise 
to  the  normal  under  proper  treatment  is  often  most 
surprising. 

When,  other  factors  remaining  unchanged,  the 
amount  of  solids  eliminated  is  considerably  above  the 
normal,  increased  destructive  metabolism  may  be 
inferred. 

The  total  solids  can  be  estimated  roughly,  but  ac- 
curately enough  for  most  clinical  purposes,  by  multi- 
plying the  last  two  figures  of  the  specific  gravity  of  the 
mixed  twenty-four-hour  urine  by  the  number  of  ounces 
voided  and  to  the  product  adding  one-tenth  of  itself. 
This  gives  the  amount  in  grains.  If,  for  example,  the 
twenty-four-hour  quantity  is  3  pints  or  48  ounces,  and 
the  specific  gravity  is  1.018,  the  total  solids  would  ap- 
proximate 950  gr.,  as  follows: 

48  X  18  =  864;   864  +  86.4  =  950.4. 

This  method  is  especially  convenient  for  the  practi- 
tioner, because  patients  nearly  always  report  the  amount 
of  urine  in  pints  and  ounces,  and  it  avoids  the  necessity 
of  converting  into  the  metric  system.  Haser's  method, 
which  uses  the  metric  system,  is  more  widely  used,  but 
is  less  convenient.  The  last  two  figures  of  the  specific 
gravity  are  multiplied  by  2.33.  The  product  is  then 
multiplied  by  the  number  of  cubic  centimeters  voided  in 
twenty-four  hours  ajid  divided  by  1000.  This  gives  the 
total  solids  in  grams. 


112  THE    URINE 

n,  FUNCTIONAL  TESTS 

Within  the  past  few  years  much  thought  has  been 
devoted  to  methods  of  more  accurately  ascertaining 
the  functional  efficiency  of  the  kidneys,  especially  of 
one  kidney  when  removal  of  the  other  is  under  con- 
sideration. The  most  promising  of  the  methods  which 
have  been  devised  are  ciyoscopy,  electric  conduc- 
tivity, the  phloridzin  test,  the  methylene-blue  test,  and 
the  phenolsulphonephthalein  test.  It  is  doubtful 
whether,  except  in  the  case  of  the  last,  these  yield  any 
more  information  than  can  be  had  from  an  intelligent 
consideration  of  the  specific  gravity  and  the  twenty- 
four-hour  quantity,  together  with  a  microscopic  exami- 
nation. They  are  most  useful  when  the  urines  obtained 
from  separate  kidneys  by  segregation  or  ureteral  cath- 
eterization are  compared.  Only  the  phenol-sulphone- 
phthalein  test  will  be  given  here.  The  reader  is 
referred  to  larger  works  upon  urinalysis  for  the  others. 
Phenolsulphonephthalein  Test.— This  test,  which  was 
offered  by  Rowntree  and  Geraghty  in  1910,  consists  in 
the  intramuscular  injection  of  a  solution  of  phenol- 
sulphonephthalein, a  drug  which  is  eliminated  only 
by  the  kidneys,  and  whose  amount  in  the  urine  is 
easily  estimated  by  colorimetric  methods.  The  time 
of  its  first  appearance  in  the  urine  and  the  quantity 
eliminated  within  a  definite  period  are  taken  as  a 
measure  of  the  functional  capacity  of  the  kidneys. 
The  test  is  harmless,  comparatively  simple,  and 
apparently  reliable.  It  will  sometimes  reveal  a  very 
serious  degree  of  renal  failure  when  twenty-four-hour 
quantity,  total  solids,  and  urea  are  practically  normal. 


FUNCTIONAL   TESTS  II3 

Technic. — The  original  procedure,  in  which  the  patient 
was  catheterized  when  the  drug  was  injected  and  the 
catheter  was  left  in  place  until  the  drug  was  detected  in  the 
urine,  is  now  seldom  followed.  The  catheter  is  still  used 
if  there  be  obstruction  to  the  outflow  of  urine  but  ordi- 
narily it  is  dispensed  with  and  the  procedure  is  as  follows: 

1.  Give  the  patient  300  to  400  c.c.  (about  2  glasses)  of 
water  to  promote  secretion  of  urine. 

2.  Twenty  minutes  afterward  have  him  empty  his 
bladder  and  discard  the  urine.  Then,  with  a  hypodermic 
syringe  inject  exactly  i  c.c.  of  the  sterile  phenolsulphone- 
phthalein  solution^  intramuscularly;  preferably  in  the 
lumbar  region. 

3.  In  exactly  one  hour  from  the  time  of  the  injection, 
have  the  patient  empty  his  bladder  and  save  all  the  urine, 

4.  In  two  hours  after  the  injection  have  the  paitient 
empty  his  bladder  again,-  and  save  all  the  urine  in  a 
separate  container.  He  should  be  under  observation 
during  the  two-hour  period,  else  it  is  difficult  to  make 
sure  that  he  carries  out  his  instructions  exactly. 

5.  Estimate  the  output  of  phenolsulphonephthalein  in 
each  of  the  two  portions  of  urine  separately  as  described 
below. 

Estimation  of  Output. — To  each  of  the  two  portions  of 
urine  add  sufficient  sodium  hydroxid  solution  to  bring  out 
the  maximum  purple-red  color.  Dilute  each  portion  to 
exactly  1000  c.c.  and  estimate  the  amount  of  the  drug 
contained  in  each  by  comparing  the  color  with  that  of  an 
alkalinized  standard  solution.  The  result  is  recorded  in 
terms  of  the  percentage  of  the  amount  injected. 

In  detail,  this  is  done  as  follows: 

1  This  solution  may  be  obtained  of  any  druggist.     It  is  sold  in  i-c.c. 
ampoules,  sterilized  ready  for  use;  but  it  should  be  noted   that  these 
ampoules  contain  somewhat  more  than  i  c.c.  hence  one  should  not 
inject  the  entire  contents. 
8 


114  THE    URINE 

1.  Add  I  c.c.  of  the  original  phenolsulphonephthalein 
solution  to  about  800  c.c.  water,  alkalinize  with  sodium 
hydroxid  and  dilute  to  1000  c.c.  Since  this  contains  the 
same  amount  of  the  drug  as  was  injected,  it  may  be  rated 
as  a  100  per  cent,  standard-color  solution.  No  more  than 
100  c.c.  of  the  standard  solution  will  be  needed,  and  there 
usually  will  be  enough  of  the  original  solution  left  in  the 
ampoule  to  make  this  amount. 

2.  Filter  the  diluted  and  alkalinized  urine  and  place 
exactly  100  c.c.  in  one  of  two  cylinder  graduates  whose 
corresponding  graduations  stand  at  the  same  height. 

3.  Into  the  other  graduate  pour  the  100  per  cent,  stand- 
ard solution,  a  little  at  a  time,  until  the  two  cylinders  show 
the  same  depth  of  color  when  looked  at  from  above  over  a 
sheet  of  white  paper.  The  height  of  the  standard  solution 
then  indicates  directly  the  excretion  percentage.  If, 
for  example,  the  color  of  the  -first  hourly  portion  was 
matched  by  40  c.c.  of  the  standard  and  that  of  the  second 
by  20  c.c,  then  the  excretion  would  be  40  per  cent,  and  20 
per  cent,  for  the  one-hour  portions  and  60  per  cent,  for 
the  two  hours. 

Another  and  probably  a  more  accurate  method  requires 
the  preparation  of  a  series  of  standard  dilutions  representing 
various  percentages  and  comparison  of  the  color  of  the 
diluted  urine  wath  these,  using  test-tubes  or  cylinders  of 
equal  diameter  and  looking  through  them  from  the  side. 
The  tubes  may  be  placed  in  an  improvised  frame  with 
ground-glass  back  like  that  of  the  Sahli  hemoglobinometer. 
The  standard  dilutions  are  easily  made:  for  the  30  per 
cent,  solution  use  30  c.c.  of  the  100  per  cent,  standard 
and  70  c.c.  water,  etc. 

In  order  to  equalize  the  slight  difference  in  color  due  to  a 
highly  colored  urine,  the  standard  color  may  be  viewed 
through  a  faintly  yellow-tinted  piece  of  glass,  or  an  amount 
of  urine  equal  to  that  voided  by  the  patient  may  be  in- 


FUNCTIONAL    TESTS  II5 

eluded  in  the  standard  solution.  For  those  who  do  much 
work  it  is  convenient  to  add  a  few  drops  of  a  solution 
of  some  yellow  dye  such  as  Echtgelb  G  or  Tropaeolin  00. 

For  greater  accuracy,  more  elaborate  colorimeters  are 
recommended.  The  simple  and  inexpensive  Denison 
Laboratory  instrument  (see  Fig.  32)  is  especially  useful  for 
this  purpose.  Results  with  this  colorimeter  are  most  de- 
pendable, when  the  unknown  solution  and  the  standard 
have  nearly  the  same  depth  of  color.  It  is  therefore  well 
to  use  a  50  per  cent,  standard  solution  for  the  phenol- 
sulphonephthalein  estimation  instead  of  the  100  per  cent, 
standard  above  recommended.  The  colorimeter  reading 
must  then  be  divided  by  two. 

When  it  is  necessary  to  defer  the  color  comparison  for 
hours  or  days,  the  urine  must  be  kept  acid  as  the  color 
fades  in  alkaline  solution. 

Under  normal  conditions  the  drug  first  appears  in 
the  urine  in  five  to  eleven  minutes  after  the  injection. 
Within  the  first  hour  40  to  60  per  cent,  is  elimi- 
nated; in  the  two  hours,  60  to  85  per  cent.  Patho- 
logically, the  excretion  may  be  reduced  to  a  trace 
or  even,  in  extreme  cases,  to  none  at  all  in  the  two 
hours. 

Time  has  proved  the  great  usefulness  of  this  test  in 
everyday  practice,  but  it  must  be  remembered  that  it  is 
a  test  of  functional  capacity  only,  not  a  measure  of  the 
extent  of  anatomic  changes  in  the  kidney.  Although 
it  is  true  that  these  generally  run  more  or  less  parallel, 
they  do  not  always  do  so.  The  test  is  extremely  valu- 
able in  diagnosis  and  prognosis  of  chronic  nephritis 
where  the  phenolsulphonephthalein  output  runs  fairly 
parallel  with  the  course  of  the  disease.  In  acute 
nephritis  the  result  does  not  always  agree  with  the 


II 6  THE    URINE 

clinical  and  pathological  picture.  Particularly  is  this 
true  in  the  acute  glomerulo-nephritis  of  scarlet  fever 
where  the  excretion  percentage  may  sometimes  be 
fully  up  to  the  normal.  Apparently  the  test  speaks 
less  definitely  concerning  glomerular  changes  than 
tubular. 

IIL  CHEMIC  EXAMINATION 

The  chemical  constituents  of  the  urine  will  be  consid- 
ered in  two  groups:  those  present  normally,  and  those 
present  in  appreciable  amount  only  under  pathologic 
conditions. 

Before  discussing  these  in  detail  it  is  convenient 
at  this  place  to  include  a  general  description  of  colori- 
metric  and  centrifugal  methods,  which  have  rather 
wide  usefulness  for  quantitative  estimations.  Their 
application  to  individual  substances  will  be  given  later. 

Colorimetric  Methods. — These  combine  comparative 
simplicity  and  great  accuracy  and  are  steadily  growing  in 
popularity. 

In  general  they  consist  in  treating  the  fluid  under  ex- 
amination with  such  reagents  as  will  produce  a  soluble 
colored  compound  with  the  substance  to  be  estimated, 
and  in  comparing  this  color  with  that  of  a  similar  solution 
of  known  strength,  upon  the  principle  that  the  depth  of 
color  is  directly  proportionate  to  the  amount  of  the  sub- 
stance present.  Some  preliminary  treatment  is  usually 
necessary  to  remove  interfering  substances.  Any  device 
which  will  show  the  quantitative  relationship  between  the 
colors  is  called  a  colorimeter. 

The  chief  hindrances  to  the  wide  adoption  of  colori- 
metric methods  for  clinical  purposes  are  the  cost  of  the 
colorimeter  and  the  difficulties  in  the  way  of  preparing 


CHEMICAL   EXAMINATION 


117 


standard  color  solutions.  Relatively  stable  standard 
solutions  for  many  of  the  methods  can  be  purchased  with 
the  instruments. 

The  Hellige  colorimeter  (Fig.  30)  is  one  of  the  most 
satisfactory    for    general   purposes.     The    solution    under 


^ 

c 

i 

Fig.  30. — Hellige  colorimeter,  i,  sliding  front,  removed;  2,  front 
view  of  interior;  3,  side  view  with  portion  of  side  wall  removed;  a,  glass 
trough  for  unknown  solution;  b,  glass  wedge  for  standard  solution; 
c,  sliding  front;  d,  window;  e,  knerled  head;/,  scale;  g,  scale-pointer; 
h,  double  prism;  i,  ground  glass  back. 

examination  is  placed  in  the  box  or  trough  {a),  while  the 
standard  solution  is  placed  in  the  wedge-shaped  bottle  (b), 
which  can  be  moved  up  or  down  beside  the  trough.  The 
front  (c)  is  slipped  into  place  and  the  two  solutions  are 


ii8 


THE    URINE 


viewed  through  the  window  (d)  behind  which  is  a  double 
prism  {h)  to  bring  the  two  colors  close  together.  The 
wedge  is  moved  up  and  down  by  means  of  the  knerled  head 
{e)  until  a  point  is  reached  where  the  two  colors  match. 
The  figure  on  the  scale  (/")  which  then  stands  opposite  the 
pointer  (g)  indicates  the  relation  between  the  strengths 
of  the  two  solutions.  If  the  pointer  stands  at  40  then  the 
unknown  solution  is  40  per  cent,  as  strong  as  the  known 
standard;  if  at  70,  it  is  70  per  cent,  as  strong.  In  the  older 
instruments  the  scale  is  reversed,  the  100  mark  being  at 
the  bottom.  In  both  types  the  actual  values  are  some- 
times found  by  reference  to  a  chart  or  "graph"  which  must 
be  made  for  each  standard  solution.  Hermetically  sealed 
standard  wedges  for  most  of  the  tests,  each  accompanied 
by  its  appropriate  graph  can  be  purchased  with  the 
instrument. 


9     10     It 

Fig.  31. — Kuttner  micro-colorimeter,  with  pipets,  graduated  tubes  and 
standard  color  tubes. 


The  Kuttner  colorimeter  (Fig.  31)  is  very  similar  to 
the  Sahli  hemoglobinometer,  the  chief  differences  being 
that  the  front  is  closed  and  the  colors  are  viewed  through  a 


CHEMICAL   EXAMINATION 


119 


window  supplied  with  a  double  prism  like  that  of  the 
Hellige.  The  unknown  solution  is  diluted  in  the  graduated 
tube  until  its  color  matches  that  of  the  standard,  which  is 
kept  in  a  sealed  tube.  The  pipets  and  test-tubes  required 
for  making  the  various  tests  are  included.     Standard  color 


1 

t 

'1 

Fig.  32. — Denison  Laboratory  colorimeter,  made  from  a  slide  box, 
blackened  inside,  and  two  30-c.c.  tubes  which  stand  upon  a  ground- 
glass  slide  and  are  held  in  place  by  a  wooden  slide. 

tubes  for  hemoglobin,   blood   sugar,   and   the  phenolsul- 
phonephthalein    kidney    test   are    now    supplied    and    the 
makers  state  that  others  are  in  preparation. 
The  Denison  Laboratory  colorimeter^  (Fig.  32)  is  prob- 

^  Designed  by  the  late  A.  R.  Peebles,  while  director  of  the  Denison 
Research  Laboratory.  Most  of  the  colorimetric  methods  which  are 
useful  in  blood  and  urine  work  have  been  modified  for  use  with  this  col- 
orimeter by  R.  C.Lewis  and  A.  R.  Peebles  and  will  be  published  soon. 


I20  THE    URINE 

ably  the  simplest,  most  convenient  and  least  expensive 
yet  devised,  and  its  accuracy  equals  or  even  exceeds  that 
of  the  Hellige  colorimeter.  The  instrument  can  be  easily 
made  by  any  one  from  a  Pillsbury  slide  box  and  two 
graduated  30-c.c.  test-tubes.  These  tubes  are  the  right 
size,  are  carried  in  stock  by  most  supply  houses,  and  an- 
swer as  well  as  specially  graduated  tubes.  Equivalent 
graduations  on  the  two  tubes  must  stand  at  the  same 
height. 

To  use  the  instrument  the  unknown  solution  is  poured 
into  one  tube  exactly  to  the  lo-c.c.  mark,  and  the  standard 
solution  is  placed  in  the  other,  a  little  at  a  time  by  means 
of  a  medicine-dropper  until  the  colors  in  the  two  tubes  just 
match  when  looked  at  from  above  over  a  sheet  of  white 
paper  or  a  small  mirror,  so  placed  that  it  reflects  the  light 
from  a  window,  A  small  reflector  can  be  placed  in  the 
bottom  of  the  box  at  an  angle  of  45  degrees  if  desired 
but  adds  to  the  cost  without  commensurate  advantage. 
When  the  two  colors  match,  the  height  of  the  standard 
color  solution — the  reading  being  taken  at  the  bottom  of 
the  meniscus — will  indicate  in  percentage  the  relation  be- 
tween the  strengths  of  the  two  solutions.  If,  for  example, 
the  top  of  the  standard  solution  stands  at  the  7.5  c.c. 
mark,  then  the  unknown  solution  is  75  per  cent,  as  strong 
as  the  known  standard.  Readings  are  most  accurate 
when  the  unknown  solution  and  the  standard  have  nearly 
the  same  depth  of  color. 

This  colorimeter  can  be  strongly  recommended  for  the 
phenolsulphonephthalein  test  (see  p.  112)  and  for  other 
estimations  when  one  prepares  his  own  standard  solutions. 

Centrifugal  Methods. — As  shown  by  Purdy,  the  cen- 
trifuge offers  a  means  of  making  quantitative  estimations 
of  a  number  of  substances  in  the  urine.  Results  are  easily 
and  quickly  obtained;  and  while  the  methods  can  lay  no 
claim  to  accuracy,  they  will  be  found  very  useful  in  follow- 


CHEMICAL   EXAMINATION 


121 


ing  the  progress  of  a  case  from  day  to  day  when  recourse  to 
more  elaborate  methods  is  out  of  the  question. 


Fig-  33- — The  Purdy  electric  centrifuge  with  four  arms. 


Fig.  34. — Water-motor  centrifuge. 


122 


THE    URINE 


In  general,  the  methods  consist  in  precipitating  the 
substance  to  be  estimated  in  a  graduated  centrifuge  tube 
by  means  of  an  appropriate  reagent,  and  applying  a  definite 
amount  of  centrifugal  force  for  a  definite  length  of  time, 
after  which  the  percentage  of  precipitate  is  read  off  upon 
the    side    of    the    tube.     Interfering    substances    such    as 


c.c\ 


Fig.  35. — Purdy's   tubes   for   the   centrifuge:  a, 
sediment  tube. 


Percentage   tube;    h. 


albumin  must  be  previously  removed.  Results  are  in 
terms  of  hulk  oj  precipitate,  which  must  not  be  confused 
with  percentage  by  weight.  The  weight  percentage  can  be 
found  by  referring  to  Purdy's  tables,  given  later;  but  in 
following  the  progress  of  the  same  case  from  day  to  day 
it  suffices  to  compare  the  bulk  of  the  precipitate,  always 
taking  into  consideration,  of  course,  the  twenty-four-hour 
amount  of  urine. 


CHEMICAL   EXAMINATION  1 23 

To  fulfil  Purdy's  requirements,  upon  which  the  tables 
are  based,  the  centrifuge  should  have  an  arm  with  a  radius 
of  6^^  inches  when  in  motion,  and  should  be  capable  of 
maintaining  a  speed  of  1500  revolutions  a  minute.  The 
electric  centrifuge  is  to  be  recommended,  although  good 
work  can  be  done  with  a  water-power  centrifuge  or,  after  a 
little  practice,  with  the  hand  centrifuge.  A  speed  indi- 
cator is  desirable  with  electric  and  water-motor  machines, 
although  one  can  learn  to  estimate  the  speed  by  the  musical 
note.  In  general  a  four-arm  centrifuge  will  be  found  most 
useful.  Instead  of  the  conical  aluminum  tube-shields 
usually  supplied,  it  is  well  to  get  flat-bottomed  shields 
with  rubber  cushions,  because  these  permit  the  use  of 
ordinary  test  tubes,  which  is  a  great  convenience  at  times. 
When  the  centrifuge  is  in  use,  opposite  tubes  must  carry 
the  same  weight,  otherwise  the  machine  will  be  quickly 
ruined.  It  is  best  to  balance  the  filled  tubes  upon  a  scale, 
but  it  will  usually  suffice  to  fill  them  to  the  same  height. 

A.    Normal  Constituents 

Of  the  large  number  of  organic  and  inorganic  sub- 
stances normally  present  in  the  urine,  only  a  few 
demand  any  consideration  from  the  clinician.  The  fol- 
lowing table,  therefore,  outlines  the  average  composition 
from  the  clinical,  rather  than  from  the  chemical, 
standpoint.  Only  the  twenty-four-hour  quantities  are 
given,  since  they  alone  furnish  an  accurate  basis  for 
comparison.  The  student  cannot  too  soon  learn  that  per- 
centages mean  little  or  nothing,  excepting  as  they  furnish 
a  means  of  caLulating  the  twenty-four-hour  elimination. 

Although  the  conjugate  sulphates  are  organic  com- 
pounds, they  are,  for  the  sake  of  convenience,  included 
with  the  inorganic  sulphates  in  the  following  table. 


124  "^^^^  URINE 

COMPOSITION  OF  NORMAL  URINE 

Grams  in  twenty- 
four  hours 

Water 1000-1500 

Total  substances  in  solution 55-70 

Inorganic  substances 20-30 

Chlorids  (chiefly  sodium  chlorid) ...  .  10-15 

Phosphates  (estimated  as  phosphoric 

acid),  total 2.5-3.5 

Earthy,  >^  of  total 

Alkaline,  %  of  total 

Sulphates     (estimated    as    sulphuric 

acid),  total i .  5-3 . o 

Mineral,  %o  of  total 

Conjugate,  3'lo  of  total 

Includes  indican 

Ammonia o .  5-1 .  o 

Organic  substances 30-40 

Urea 25-35 

Uric  acid o .  4-1 .  o 


Approximate 

average 

1200 

60 

25 

12. 

5 

3 

I 

2 

2. 

5 

2. 

25 

0. 

25 

Trace 

0. 

7 

35 

30 

0. 

■7 

Among  constituents  which  are  of  little  clinical  im- 
portance, or  are  present  only  in  traces,  are: 

Inorganic. — Iron,  carbonates,  nitrates,  silicates,  and 
fluorlds. 

Organic. — Creatinin,  hippuric  acid,  purin  bases,  oxalic 
acid,  volatile  fatty  acids,  pigments,  and  acetone. 

Variations  in  body  weight,  diet,  and  exercise  cause 
marked  fluctuations  in  the  total  solids  and  in  individual 
substances. 

1.  Chlorids.— These  are  derived  from  the  food,  and 
are  mainly  in  the  form  of  sodium  chlorid.  The  amount 
excreted  normally  is  10  to  15  Gm.  in  twenty-four  hours. 
It  is  much  afifected  by  the  diet,  and  is  reduced  to  a  mini- 
mum in  starvation. 

Excretion  of  chlorids  is  diminished  in  nephritis  and 


CHEMICAL  EXAMINATION 


125 


Fig.  36. — Graphic  expression  of  quantities  in  the  urine.  Solid  line, 
normal  urine;  dotted  line,  an  example  of  pathologic  urine  in  a  case  of 
cancerous  cachexia  (Saxe). 


126  THE    URINE 

in  fevers,  especially  in  pneumonia  and  inflammations 
leading  to  the  formation  of  large  exudates.  In  nephritis 
the  kidneys  are  less  permeable  to  the  chlorids,  and  it  is 
possible  that  the  edema  is  due  largely  to  an  effort  of 
the  body  to  dilute  the  chlorids  which  have  been  retained. 
Certainly  an  excess  of  chlorids  in  the  food  will  in  many 
cases  increase  both  the  albuminuria  and  the  edema  of 
nephritis.  In  fevers  the  diminution  is  due  largely  to  de- 
crease of  food,  though  probably  in  some  measure  to 
impaired  renal  function.  In  pneumonia  chlorids  are 
constantly  very  low,  and  in  some  cases  are  absent  en- 
tirely. Following  the  crisis  they  are  increased.  In  in- 
flammations leading  to  formation  of  large  exudates — e.g., 
pleurisy  with  efl'usion — chlorids  are  diminished  because 
a  considerable  amount  becomes  "locked  up"  in  the 
exudate.  During  absorption  chlorids  are  liberated  and 
appear  in  the  urine  in  excessive  amounts. 

Diminution  of  chlorids  is  also  sometimes  observed  in 
severe  diarrhea,  anemic  conditions,  and  carcinoma  of 
the  stomach. 

Detection  of  Chlorids. — The  following  simple  test  will 
show  the.  presence  of  chlorids,  and  at  the  same  time 
roughly  indicate  any  pronounced  alteration  in  amount: 

To  a  few  cubic  centimeters  of  urine  in  a  test-tube  add  a 
few  drops  of  nitric  acid  to  prevent  precipitation  of  phos- 
phates and  then  a  few  drops  of  silver  nitrate  solution  of 
about  12  per  cent,  strength.  A  white,  curdy  precipitate 
of  silver  chlorid  forms.  If  the  urine  merely  becomes 
milky  or  opalescent,  chlorids  are  markedly  diminished. 

Quantitative  Estimation. — The  well-known  and  re- 
liable Volhard  method  has  been  simplified  by  Strauss, 
and  this  modification  has  recently  been  still  further 


CHEMICAL   EXAMINATION  127 

simplified  by  Bayne- Jones  and  by  McLean  and  Selling, 
so  that  the  method  is  now  available  for  ordinary  clin- 
ical work .  The  only  difficulty  is  the  preparation  of  solu- 
tions, and  these  can  be  purchased  ready  prepared.  A 
much  less  accurate,  though  simple  and  very  useful, 
method  is  afforded  by  the  centrifuge. 

I.  Simplified  Volhard  Method. — As  a  rule  albumin  need 
not  be  removed.  In  an  accurately  graduated  50-c.c. 
cylinder  place  5  c.c.  of  the  urine  and  10  c.c.  of  Solution 
No.  I.  Mix  by  inverting  several  times.  If  a  reddish  color 
appears,  add  3  drops  of  10  per  cent,  potassium  perman- 
ganate. After  five  minutes  add  Solution  No.  2,  a  very 
little  at  a  time,  mixing  after  each  addition,  until  a  perma- 
nent red-brown  color  (best  seen  against  a  white  back- 
ground) appears.     This  is  the  end-point. 

The  solutions  are  so  balanced  that  if  the  urine  be  chlorid- 
free  the  volume  of  fluid  when  the  end-point  is  reached  will 
be  35  c.c,  and  that  for  each  gram  per  Uter  of  chlorids  in 
the  urine  the  volume  will  be  i  c.c.  less.  Therefore,  the  dif- 
ference between  35  c.c.  and  the  height  of  the  fluid  at  the 
end  of  the  test  gives  directly  the  number  of  grams  of  chlo- 
rids per  liter  of  urine,  expressed  as  sodium  chlorid.  If,  for 
example,  the  fluid  reaches  the  28-c.c.  mark,  35  —  28  =  7 
Gm.  of  sodium  chlorid  per  liter  of  urine. 

A  certified  50-c.c.  graduated  cylinder,  with  glass  stopper, 
is  required.     The  ordinary  50-c.c.  graduate  is  inaccurate. 

The  solutions  are  as  follows: 

No.  I. — Standard  silver  nitrate  solution: 

Silver  nitrate  (C. P.,  anhydrous,  crystallized).       29.055  Gm.; 

Nitric  acid  (25  per  cent.) 900  c.c; 

Ammonioferric   alum    (cold   saturated   solu- 
tion)         50  c.c. 

Distilled  water  to 1000  c.c. 


128 


THE    URINE 


No.  2, — Ammonium  sulphocyanate  solution: 

Ammonium  sulphocyanate 7  Gm.; 

Distilled  water 1000  c.c. 

This  solution  is  intentionally  made  too  strong,  and  it  must  be 
standardized  by  diluting  with  distilled  water  until  exactly  20  c.c. 
(and  no  less)  will  produce  a  red  color  when  mixed  with  exactly 
10  c.c.  of  Solution  No.  i. 

TABLE  FOR  THE  ESTIMATION  OF  CHLORIDS  AFTER 
CENTRIFUGATION 

Showing  the  bulk- percentage  of  silver  chlorid  {AgCl)  and  the  correspond- 
ing gravimetric  percentages  sodium  chlorid  {NaCl)  and  chlorin  (CI). — 
{Purdy.) 


Bulk- 
percentage 
of  AgCl. 

Percentage 
NaCl. 

Percentage 
CI. 

Bulk- 
percentage 
of   AgCl. 

Percentage 
NaCl. 

Percentage 
CI. 

K 

0.03 

0.02 

8 

1.04 

0.63 

M 

0.07 

0.04 

8>^ 

I  .1 

0.67 

M 

0. 1 

0.06 

9 

I. 17 

0.71 

I 

0.13 

0.08 

gVz 

1.23 

0.75 

iM 

0.16 

O.I 

10 

1-3 

0.79 

iM 

0.19 

0.12 

loM 

1.36 

0.83 

xV^ 

0.23 

0. 14 

II 

1-43 

0.87 

2 

0.26 

0.16 

iiM 

1.49 

0.91 

2M 

0.29 

0.18 

12 

1-5° 

0-95 

2>r 

0.32 

0.  2 

^2y^ 

1 .62 

0.99 

2% 

0.36 

0.  22 

13 

1 .69 

1.02 

3 

0-39 

0.  24 

nVi 

1-75 

1.06 

3K 

0.42 

0.26 

14 

1.82 

1. 1 

3M 

0.4s 

0.28 

taM 

1.88 

1. 14 

3M 

0.49 

03 

15 

1.94 

1. 18 

4 

0.52 

0.32 

I5>'2 

2.01 

1.22 

A\i 

0.5s 

0.34 

16 

2.07 

1.26 

4M 

0.58 

0.3s 

I6M 

2.14 

1-3 

4^i 

0.62 

0-37 

17 

2.2 

1-34 

5 

0.65 

0-39 

17H 

2.  27 

1.38 

sVi 

0.71 

0-43 

18 

2-33 

1.42 

6 

0.78 

0.47 

I8M 

2.4 

1.46 

(>V2 

0.84 

0.51 

19 

2.46 

i-S 

7 

0.91 

o-SS 

i9>^ 

2.53 

1-54 

7M 

0.97 

OS9 

20 

2-59 

1.58 

Bulk-percentage  to  be  read  on  the  side  of  the  tube. 


CHEMICAL   EXAMINATION  1 29 

2.  Centrifugal  Method. — Fill  the  graduated  tube  to  the 
lo-c.c.  mark  with  urine;  add  15  drops  strong  nitric  acid  and 
then  silver  nitrate  solution  of  12  per  cent,  strength  to  the 
15-C.C.  mark.  Mix  by  inverting  several  times.  Let  stand  a 
few  minutes  for  a  precipitate  to  form,  and  then  revolve  in  the 
centrifuge  for  three  minutes  at  1200  revolutions  a  minute. 
Each  0.1  c.c.  of  precipitate  equals  i  per  cent,  by  bulk.  This 
may  be  converted  into  percentage  by  weight  of  chlorin  or 
sodium  chlorid  by  means  of  the  table  upon  page  128. 

2.  Phosphates  are  derived  largely  from  the  food, 
only  a  small  proportion  resulting  from  metabolism. 
The  normal  daily  output  of  phosphoric  acid  is  about  2.5 
to  3.5  Gm.* 

The  urinary  phosphates  are  of  two  kinds:  alkaline, 
which  make  up  two-thirds  of  the  whole,  and  include  the 
phosphates  of  sodium  and  potassium ;  and  earthy,  which 
constitute  one-third,  and  include  the  phosphates  of  cal- 
cium and  magnesium.  Earthy  phosphates  are  fre- 
quently thrown  out  of  solution  in  neutral  and  alkaline 
urines,  and  as  "amorphous  phosphates"  form  a  very 
common  sediment.  This  sediment  seldom  indicates  an 
excessive  excretion  of  phosphoric  acid.  It  is  usually 
merely  an  evidence  of  diminished  acidity  of  the  urine,  or 
of  an  increase  in  the  proportion  of  phosphoric  acid  elimi- 
nated as  earthy  phosphates.  This  form  of  "phosphat- 
uria"  is  most  frequent  in  neurasthenia  and  hysteria. 
When  the  urine  undergoes  ammoniacal  decomposition, 
some  of  the  ammonia  set  free  combines  with  magnesium 
phosphate  to  form  ammoniomagnesium  phosphate 
("triple  phosphate"),  which  is  only  slightly  soluble  in 
alkaline  urine  and  is  deposited  in  tj^ical  crystalline 
form  (see  p.  211). 


I30 


THE    URINE 


Excretion  of  phosphates  is  increased  by  a  diet  rich  in 
nucleins;  in  active  metabohsm;  in  certain  nervous  and 
mental  disorders;  in  leukemia;  and  in  phosphatic  dia- 
betes, an  obscure  disturbance  of  metabolism  (not  related 
to  diabetes  mellitus)  which  is  associated  with  an  increase 
in  the  output  of  phosphates  up  to  lo  Gm.  or  more  in 
twenty-four  hours.  Phosphates  are  decreased  in  chronic 
diseases  with  lowered  metabolism;  in  hepatic  cirrhosis 
and  acute  yellow  atrophy;  in  pregnancy,  owing  to  de- 
veloping fetal  bones;  and  in  nephritis,  owing  to  kidney 
impermeability. 


TABLE  FOR  THE  ESTIMATION  OF  PHOSPHATES  AFTER 
CENTRIFUGATION 

Showing  bidk- percentages  of  uranyl  phosphate  iH[U02]P0i)  and  the 
corresponding  g/avimetric  percentages  of  phosphoric  acid  {PjO^). — 
(Purdy.) 


Bulk-percentage 
of  H(U02)P0«. 

Percentage 

Pl06. 

Bulk-percentage 
of  H(U02)P0<. 

Percentage 
P2OS. 

M 

0.02 

1                 11 

0.  14 

I 

0.04 

12 

015 

l>^ 

0.045 

13 

0.  16 

2 

0.05 

14 

0.17 

2M 

o-oss 

15 

0.18 

3 

0.06 

16 

0.  19 

3M 

0.065 

17 

0.  2 

4 

0.07 

18 

0.  21 

4M 

0.075 

19 

0.  22 

5 

0.08 

20 

0.23 

6 

o.og 

21 

0.  24 

7 

0. 1 

22 

0.25 

8 

0.  II 

23 

0.26 

9 

0.12 

24 

0.27 

lO 

0.13 

25 

0.28 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 


CHEMICAL   EXAMINATION  I31 

Quantitative  estimation  does  not  furnish  much  of 
definite  clinical  value.  The  centrifugal  method  is  the 
most  convenient. 

Purdy's  Centrifugal  Method. — Take  lo  c.c.  urine  in  the 
graduated  tube,  add  2  c.c.  of  50  per  cent,  acetic  acid,  and 
3  p.c.  of  5  per  cent,  uranium  nitrate  solution.  Mix;  let 
stand  a  few  minutes,  and  revolve  for  three  minutes  at 
1200  revolutions  a  minute.  Each  o.i  c.c.  of  precipitate  is 
I  per  cent,  by  bulk.  The  corresponding  percentage  of 
phosphoric  acid  by  weight  is  found  by  consulting  the  table 
on  page  130. 

3.  Sulphates. — The  urinary  sulphates  are  derived 
partly  from  the  food,  especially  meats,  and  partly  from 
body  metabolism.  The  normal  output  of  sulphuric  acid 
is  about  1.5  to  3  Gm.  daily.  It  is  increased  in  condi- 
tions associated  with  active  metabolism,  and  in  general 
may  be  taken  as  a  rough  index  of  protein  metabolism. 

Quantitative  estimation  of  the  total  sulphates  yields 
little  of  clinical  value. 

Purdy's  Centrifugal  Method. — Take  10  c.c.  urine  in  the 
graduated  tube  and  add  5  c.c.  barium  chlorid  solution 
(barium  chlorid,  4  parts;  concentrated  hydrochloric  acid, 
I  part;  and  distilled  water,  16  parts).  Mix;  let  stand  a 
few  minutes,  and  revolve  for  three  minutes  at  1200  revo- 
lutions a  minute.  Each  o.i  c.c.  of  precipitate  is  i  per 
cent,  by  bulk.  The  percentage  by  weight  of  sulphuric 
acid  is  calculated  from  the  table  on  page  132. 

About  nine- tenths  of  the  sulphuric  acid  is  in  com- 
bination with  various  mineral  substances,  chiefly  sodium, 
potassium,  calcium,  and  magnesium  (mineral  or  pre- 
formed sulphates).     One-tenth  is  in  combination  with 


132 


THE   URINE 


certain  aromatic  substances,  which  are  mostly  products 
of  protein  putrefaction  in  the  intestine,  but  are  de- 
rived in  part  from  destructive  metabolism  {conjugate 
or  ethereal  sulphates) .  Among  these  aromatic  substances 
are  indol,  phenol,  and  skatol.  By  far  the  most  impor- 
tant of  the  conjugate  sulphates  and  representative  of 
the  group  is  potassium  indoxyl  sulphate. 

* 
TABLE  FOR  THE  ESTIMATION  OF  SULPHATES  AFTER 
CENTRIFUGATION 

ShoTving  the  bulk-percentages  of  barium  sulphate  {BaSO^  and  the  cor- 
responding gravimetric  percentages  of  sulphuric  acid  (SOj). — {Purdy.) 


BuUc-percentr  ge 

Percentage          1 

Bulk-percentage 

Percentage 

of  BaSOi. 

1 

so^  ■ 

of  BaSO«. 

SOs. 

H 

0.04 

2H 

o-SS 

H 

0.07 

2H 

0.61 

H 

O.I 

2^ 

0.67 

M 

0  13 

3 

0-73 

H 

0.16 

sH 

0  79 

y* 

0. 19 

sH 

0.85 

H 

0.22 

sH 

0.91 

I 

0.25 

4 

0.97 

iM 

0.31 

aH 

I   03 

iM 

0-37 

4H 

-       1.09 

iV* 

0.43 

4H 

115 

2 

0.49 

5 

I   21 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 


Potassium  indoxyl  sulphate,  or  indican,  is  derived 
from  indol.  Indol  is  absorbed  and  oxidized  into  in- 
doxyl, which  combines  with  sulphuric  acid  and  potas- 
sium and  is  thus  excreted.  Under  normal  conditions 
the  amount  in  the  urine  is  small.  It  is  increased  by  a 
meat  diet. 


CHEMICAL   EXAAONATION  I33 

Unlike  the  other  ethereal  sulphates,  which  are  de- 
rived in  part  from  metaboUsm,  indican  originates  prac- 
tically wholly  from  putrefactive  processes.  It  alone, 
therefore,  and  not  the  total  ethereal  sulphates,  can  be 
taken  as  an  index  of  such  putrefaction.  A  pathologic 
increase  is  called  indicanuria.  It  is  noted  in: 
■  (a)  Diseases  of  the  Small  Intestine. — This  is  by  far 
the  most  common  source.  Intestinal  obstruction  gives 
the  largest  amounts  of  indican.  It  is  also  much  in- 
creased in  intestinal  indigestion — so-called  "  bilious- 
ness;" in  inflammations,  especially  in  cholera  and  ty- 
phoid fever;  and  in  paralysis  of  p)eristalsis,  such  as 
occurs  in  peritonitis.  Simple  constipation  and  diseases 
of  the  large  intestine  alone  do  not  so  frequentiy  cause 
indicanuria. 

(b)  Diseases  of  the  stomach  associated  with  deficient 
hydrochloric  acid,  as  chronic  gastritis  and  gastric  cancer. 
Diminished  hydrochloric  add  favors  intestinal  putre- 
faction. 

(c)  Diminished  Flow  of  Bile. — Since  the  bile  serves 
as  a  stimulant  to  peristalsis  and  in  several  ways  re- 
tards putrefaction,  a  diminished  flow  from  any  cause 
favors  occurrence  of  indicanuria. 

(d)  Decompositian  of  exudates  anywhere  in  the  body, 
as  in  empyema,  bronchiectasis,  and  large  tuberculous 
ca\-ities. 

Detection  of  indican  depends  upon  its  decomposition 
and  oxidation  of  the  indoxyl  set  free  into  indigo-blue. 
This  change  sometimes  takes  place  spontaneously  in 
decomposing  urine,  causing  a  dirty  blue  color.  Crystals 
of  indigo  (see  Fig.  49)  may  then  be  fotlnd  both  in  the 
sediment  and  the  scum. 


134  THE   URINE 

Obermayer's  Method. — Take  a  test-tube  about  one- 
third  full  of  the  urine  and  add  an  equal  volume  of  Ober- 
mayer's reagent  and  a  few  cubic  centimeters  of  chloroform. 
Mix  by  inverting  a  few  times;  avoid  shaking  violently. 
If  indican  be  present  in  excess,  the  chloroform,  which  sinks 
to  the  bottom,  will  assume  an  indigo-blue  color.  It  will 
take  up  the  indigo  more  quickly  if  the  urine  be  warm. 
The  depth  of  color  indicates  the  comparative  amount  of 
indican  if  the  same  proportions  of  urine  and  reagents  are 
always  used,  but  one  should  bear  in  mind  the  total  amount 
of  urine  voided.  The  indican  in  normal  urine  may  give  a 
faint  blue  by  this  method.  Urine  of  patients  taking  iodids 
gives  a  reddish-violet  color,  which  disappears  upon  addi- 
tion of  a  few  drops  of  strong  sodium  hyposulphite  solution 
and  shaking.  Occasionally,  owing  to  slow  oxidation,  indigo- 
red  will  form  instead  of  indigo-blue.  This  gives  a  color  like 
that  due  to  iodids,  but  it  does  not  disappear  when  treated 
with  sodium  hjqjosulphite.  Bile-pigments,  which  inter- 
fere with  the  test,  must  be  removed  if  present  (see  p.  loi). 

Obermayer's  reagent  consists  of  strong  hydrochloric  acid 
(sp.  gr.,  1. 19),  1000  c.c,  and  ferric  chlorid,  2  Gm.  This 
makes  a  yellow,  fuming  liquid  which  keeps  well. 

4.  Urea. — From  the  standpoint  of  physiology  urea  is 
the  most  important  constituent  of  the  urine.  It  is  the 
principal  waste-product  of  metabolism,  and  constitutes 
about  one-half  of  all  the  solids  excreted — about  20  to 
35  Gm.  in  twenty-four  hours.  It  represents  85  to  90 
per  cent,  of  the  total  nitrogen  of  the  urine,  and  its  quan- 
titative estimation  is  a  simple,  though  not  very  accurate, 
method  of  ascertaining  the  state  of  nitrogenous  excretion. 
This  is  true,  however,  only  in  normal  individuals 
upon  average  Aixed  diet.  Upon  a  low  protein  diet  it 
may  fall  to  60  per  cent,  of  the  total  nitrogen.     Under 


CHEMICAL   EXAMINATION  I35 

pathologic  conditions,  tfie  proportion  of  nitrogen 
distributed  among  the  various  nitrogen-containing 
substances  undergoes  great  variation.  The  only  ac- 
curate index  of  protein  metabolism  is,  therefore,  the 
total  output  of  nitrogen,  which  can  be  estimated  by  the 
Kjeldahl  method  or  one  of  its  modifications  such  as  the 
new  direct  Nesslerization  method  of  Folin  and  Denis. 
The  whole  subject  of  "nitrogen  partition"  (distribution 
of  nitrogen  among  the  nitrogen-containing  bodies)  and 
''nitrogen  equihbrium"  (relation  of  excretion  to  intake) 
is  an  important  one,  but  is  out  of  the  province  of  this 
book,  since  as  yet  it  concerns  the  physiologic  chemist 
more  than  the  clinician. 

It  may  be  helpful  to  state  here,  however,  that  upon  a 
mixed  diet  the  nitrogen  of  the  urine  is  distributed  about  as 
follows:  urea  nitrogen,  86.9  per  cent.;  ammonia  nitrogen, 
4.4  per  cent.;  creatinin  nitrogen,  3.6  per  cent.;  uric  acid 
nitrogen,  0.75  per  cent.;  "undetermined  nitrogen,"  chiefly 
in  amino-acids,  4.3  per  cent. 

Normally,  the  amount  is  greatly  influenced  by  ex- 
ercise and  diet.  It  is  increased  by  copious  drinking 
of  water  and  administration  of  ammonium  salts  of 
organic  acids. 

Pathologically,  urea  is  increased  in  fevers,  in  diabetes 
when  acidosis  is  not  marked,  and  especially  during 
resolution  of  pneumonia  and  absorption  of  large  exu- 
dates. As  above  indicated,  when  other  factors  are 
equal,  the  amount  of  urea  indicates  the  activity  of 
metabolism.  In  deciding  whether  in  a  given  case  an 
increase  of  urea  is  due  to  increased  metabolism  the 
relation  between  the  amounts  of  urea  and  of  the  chlorids 


136  THE    URINE 

is  a  helpful  consideration.  Upon  a  mixed  diet  the 
amount  of  urea  is  normally  about  twice  that  of  the 
chlorids.  If  the  proportion  is  much  increased  above 
this,  increased  tissue  destruction  may  be  inferred,  since 
other  conditions  which  increase  urea  also  increase 
chlorids. 

In  general,  a  pathologic  decrease  in  amount  of  urea 
is  due  either  to  lessened  formation  within  the  body  or 
to  diminished  excretion.  Decreased  formation  of  urea 
occurs  in  diseases  of  the  liver  with  destruction  of  liver 
substance,  such  as  marked  cirrhosis,  carcinoma,  and 
acute  yellow  atrophy.  The  state  of  acidosis  likewise 
decreases  formation  of  urea,  because  nitrogen  which 
would  otherwise  be  built  into  urea  is  eliminated  in  the 
form  of  ammonia  (see  p.  145).  Retention  of  urea  occurs 
in  most  cases  of  nephritis.  In  acute  nephritis  the 
amount  of  urea  in  the  urine  is  markedly  decreased, 
and  a  return  to  normal  denotes  improvement.  In  the 
early  stages  of  chronic  nephritis,  when  diagnosis  is 
difficult,  it  is  usually  normal.  In  the  late  stages,  when 
diagnosis  is  comparatively  easy,  it  is  decreased.  Hence 
estimation  of  urea  is  of  Uttle  help  in  the  diagnosis  of 
this  disease,  and  is  of  no  value  whatever  when,  as 
is  so  frequently  the  case,  a  small  quantity  of  urine 
taken  at  random  is  used.  When,  however,  the  diag- 
nosis is  established,  estimations  made  at  frequent  in- 
tervals under  the  same  conditions  of  diet  and  exercise 
are  of  much  value,  provided  a  sample  oj  the  mixed  twenty- 
four-hour  urine  be  used.  A  steady  decline  is  a  very 
bad  prognostic  sign,  and  a  sudden  marked  diminution 
is  usually  a  forerunner  of  uremia. 

The  presence  of  urea  can  be  shown  by  allowing  a  few 


CHEMICAL   EXAMESTATION 


137 


drops  of  the  fluid  partially  to  evaporate  upon  a  slide, 
and  adding  a  small  drop  of  pure,  colorless  nitric  acid  or 
saturated  solution  of  oxalic  acid.  Crystals  of  urea 
nitrate  or  oxalate  (Fig.  37)  will  soon  appear  and  can  be 
recognized  with  the  microscope. 


Fig.  37. — Crystals  of  nitrate  of  urea 
(upper  half)  and  oxalate  of  urea  (lower 
half)  (after  Funke). 


Fig,  38.  —  Doremus- 
Hinds'  ureometer  with- 
out foot. 


Quantitative  Estimation. — The  hypobromite  method, 
which  has  long  been  used  in  clinical  work,  is  very  simple, 
but  is  notoriously  inaccurate.  The  new  urease  methods 
are  much  more  accurate. 

I.  Hypobromite  Method. — This  depends  upon  the  fact 
that  urea  is  decomposed  by  sodium  hypobromite  with 
liberation  of  nitrogen.  The  amount  of  urea  is  calculated 
from  the  volume  of  nitrogen  set  free.  Of  the  many  forms 
of  apparatus  devised  for  this  purpose,  that  of  Doremus- 
Hinds  (Fig.  38)  is  probably  the  most  convenient. 

Pour  some  of  the  urine  into  the  smaller  tube  of  the  appa- 


138  THE   URINE 

ratus,  then  open  the  stop-cock  and  quickly  close  it  so  as  to 
fill  its  lumen  with  urine.  Rinse  .out  the  larger  tube  with 
water  and  fill  it  and  one-half  of  the  bulb  with  25  per  cent, 
caustic  soda  solution.  Add  to  this  i  c.c.  of  bromin  by 
means  of  a  medicine-dropper  and  mix  well.  This  prepares 
a  fresh  solution  of  sodium  hypobromite  with  excess  of 
caustic  soda,  which  serves  to  absorb  the  carbon  dioxid 
set  free  in  the  decomposition  of  urea.  When  handling 
bromin,  keep  an  open  vessel  of  ammonia  near  to  neutralize 
the  irritant  fumes. 

Pour  the  urine  into  the  smaller  tube,  and  then  turn  the 
stop-cock  so  as  to  let  as  much  urine  as  desired  (usually 
I  c.c.)  run  slowly  into  the  hypobromite  solution.  When 
bubbles  have  ceased  to  rise,  read  off  the  height  of  the  fluid 
in  the  large  tube  by  the  graduations  upon  its  side.  This 
gives  the  amount  by  weight  of  urea  in  the  urine  added, 
from  which  the  amount  excreted  in  twenty-four  hours  can 
easily  be  calculated.  If  the  urine  contains  much  more 
than  the  normal  amount,  it  should  be  diluted. 

This  method  has  fallen  into  disrepute  largely  because  of 
inconstant  results,  and  because  it  gives  more  nearly  the  total 
nitrogen  than  the  urea.  According  to  Robinson  and 
Miiller  the  discrepancies  are  due  to  insufficient  mixing  of 
urine  and  hypobromite  and  can  be  obviated  by  gentle 
shaking  after  the  first  vigorous  reaction  is  over.  Results 
are  then  constant,  but  too  high,  owing  to  decomposition 
of  other  nitrogenous  constituents. 

To  avoid  handling  pure  bromin,  which  is  disagreeable, 
Rice's  solutions  may  be  employed: 

(a)  Bromin 31  Gm.; 

Potassium  bromid 31  Gm.; 

Distilled  water 250  c.c; 

(6)  Sodium  hydroxid 100  Gm. ; 

Distilled  water 250  c.c. 


CHEMICAL   EXAMINATION  1 39 

Equal  parts  of  these  solutions  are  mixed  and  used  for  the 
test.  The  bromin  solution  must  be  kept  in  a  tightly  stop- 
pered bottle  or  it  will  rapidly  lose  strength. 

2.  Urease  Methods. — ^These  are  based  upon  the  con- 
version of  urea  into  ammonium  carbonate  by  urease,  a 
ferment  first  extracted  by  Takeuchi  from  the  soy  bean  in 
1909.  The  urea  is  estimated  from  the  amount  of  am- 
monium carbonate  produced  by  the  fermentation.  There 
are  several  clinical  methods,  two  of  which  are  here  given 
in  detail.  In  the  first,  the  urine  after  fermentation  is 
titrated  with  decinormal  hydrochloric  acid  in  the  presence 
of  the  indicator,  methyl  orange.  It  is  not  entirely  accurate, 
but  is  much  superior  to  the  hj^obromite  method.  In  the 
second,  the  ammonia  is  determined  by  direct  Nesslerization. 
This  method  is  sufficiently  accurate  for  the  most  exacting 
work  but  is  too  complicated  for  use  in  a  physician's  office 
laboratory.  It  is  included  here  because  of  the  growing 
importance  of  exact  estimations  of  the  various  nitrogenous 
substances  in  blood  and  urine. 

Neither  albumin  nor  sugar  nor  any  other  substance 
likely  to  be  present  in  body  fluids  interferes  with  the 
action  of  urease. 

Marshall's  Urease  Method. — i.  Into  each  of  two  200-c.c. 
flasks  measure  5  c.c.  of  the  urine  and  about  100  c.c.  of 
water,  and  to  one  add  i  c.c.  of  a  10  per  cent,  solution  of 
urease.^ 

2.  Overlay  the  fluid  in  each  flask  with  about  i  c.c.  of 
toluol,  insert  corks  and  let  stand  over  night  at  room  tem- 
perature (or  for  three  hours  in  the  incubator  at  37°C.). 

3.  At  the  end  of  this  time,  titrate  the  contents  of  each 
flask  to  a  distinct  pink  color  with  decinormal  hydrochloric 

^  It  is  more  convenient  to  use  the  0.025-Gm.  tablets  sold  byHynson, 
Westcott  and  Dunning,  Baltimore.  One  of  these  is  crushed  and  dis- 
solved in  5  c.c.  of  water,  and  the  whole  of  this  solution  is  used  for  the 
test. 


I40  THE    URINE 

acid,  using  a  few  drops  of  0.5  per  cent,  methyl  orange  solu- 
tion as  indicator. 

4.  Find  the  difference  between  the  number  of  cubic 
centimeters  of  decinormal  acid  used  in  the  two  titrations 
and  multiply  this  by  the  factor  0.06  to  obtain  the  per- 
centage of  urea  in  the  urine.  From  the  percentage  calculate 
the  twenty-four-hour  elimination. 

Urease  Method  of  Folin  and  Denis. — This  method  is 
comparatively  simple  if  one  has  the  reagents  at  hand.  All 
except  the  soy  bean  meal  suspension  can  be  purchased  ready 
prepared,  and  for  this,  the  tablets  mentioned  in  the  foot-note 
on  page  139  may  be  substituted.  Ammonia-free  distilled 
water  must  be  used  throughout. 

Reagents  Required. — (a)  One  per  cent,  suspension  of  soy 
bean  flour.  The  "soja  bean  meal"  which  is  sold  as  a 
food  for  diabetics  and  is  obtainable  from  any  wholesale 
drug  house  may  be  used.  Rub  up  5  Gm.  of  the  meal  to 
a  uniform  paste  with  about  15  c.c.  water,  and  gradually 
add  enough  water  to  make  400  c.c.  To  this  add  100  c.c. 
alcohol.  The  suspension  remains  good  for  about  two 
days. 

{b)  Nessler's  reagent.  Dissolve  7.5  Gm.  of  potassium 
iodid  in  50  c.c.  warm  water  and  add  10  Gm.  of  mercuric 
iodid.  Add  about  40  c.c.  water,  filter  and  dilute  to  100  c.c. 
This  is  a  stock  solution.  The  Nessler's  solution  to  be  used 
in  this  method,  consists  of  30  c.c.  of  the  stock  solution,  20 
c.c.  of  10  per  cent,  sodium  hydroxid  and  50  c.c.  water. 

(c)  Standard  ammonium  sulphate  solution.  In  exactly 
1000  c.c.  distilled  water  dissolve  4.716  Gm.  Kahlbaum's^ 
C.P.  ammonium  sulphate  which  has  been  dried  for  an 
hour  at  iio°C.  before  weighing.  Take  10  c.c.  of  this 
solution   and   dilute   to   exactly   200   c.c.     Twenty   cubic 

^  Most  ammonium  sulphate  contains  pyridine  bodies  which  interfere 
with  the  Nesslerization,  In  the  future  it  should  be  possible  to  ob- 
tain satisfactory  ammonium  sulphate  of  American  make. 


CHEMICAL   EXAMINATION  141 

centimeters  of  this  final  solution  contains  exactly  0.00 1 
Gm.  of  nitrogen. 

(d)  Meta-phosphoric  acid,  25  per  cent,  aqueous  solution 
made  without  heat.  This  deteriorates  after  two  or  three 
days. 

(e)  Merck's  blood  charcoal. 

Method. — I.  Place  exactly  i  c.c.  of  the  urine  in  a  loo-c.c. 
flask,  add  10  or  15  c.c.  of  the  i  per  cent,  soy  meal  suspension, 
stopper  the  flask  and  let  stand  for  one  hour  at  room  tem- 
perature or  fifteen  minutes  in  a  water  bath  at  5o°C. 

2.  Add  25  c.c.  water  and  i  c.c.  fresh  25  per  cent,  meta- 
phosphoric  acid.     Mix  well. 

3.  Add  about  i  Gm.  Merck's  blood  charcoal,  make 
up  to  100  c.c.  and  filter. 

4.  To  10  c.c.  of  the  filtrate  add  about  60  c.c.  distilled 
water  and  15  c.c.  Nessler's  solution,  and  make  up  to  100 
c.c.  with  distilled  water. 

5.  Make  up  a  standard  solution  representing  o.ooi  Gm. 
ammonia  nitrogen  as  follows:  To  20  c.c.  of  the  standard 
ammonium  sulphate  solution  add  15  c.c.  Nessler's  solution, 
and  make  up  to  100  c.c.  with  distilled  water. 

6.  Find  the  strength  of  the  unknown  solution  by  com- 
paring it  with  the  standard  in  a  colorimeter.  The  quantity 
of  nitrogen  in  the  unknown  represents  the  urea-nitrogen 
plus  the  ammonia  nitrogen  in  o.i  c.c.  urine,  and  must  be 
multiplied  by  1000  to  find  the  amount  in  100  c.c.  urine. 
If,  for  example,  the  reading  on  one  of  the  newer  Hellige 
instruments  is  70,  then  the  unknown  solution  (representing 
0.1  c.c.  urine)  contains  0.0007  G"^-  combined  urea  and 
ammonia  nitrogen,  and  100  c.c.  urine  contains  0.7  Gm. 

7.  In  another  sample  of  urine  estimate  ammonia  nitrogen 
alone  for  100  c.c.  urine  (see  p.  147)  and  subtract  this  from 
the  figure  obtained  above.  The  remainder  is  the  urea 
nitrogen  for  100  c.c.  of  urine.  To  express  this  in  terms  of 
urea  multiply  by  2.14. 


142  THE   URINE 

This  method,  slightly  modified,  is  also  applicable  to 
estimation  of  urea  in  blood,  ammonia  in  urine  (see  p.  147) 
and  total  nitrogen  in  urine. 

5.  Uric  acid  is  the  most  important  of  a  group  of 
substances,  called  purin  bodies,  which  are  derived  chiefly 
from  the  nucleins  of  the  food,  exogenous  uric  acid,  and 
from  metabolic  destruction  of  the  nuclei  of  the  body, 
endogenous  uric  CLcid.  The  daily  output  of  uric  acid  is 
about  0.4  to  I  Gm.  The  amount  of  the  other  purin 
bodies  together  is  about  one- tenth  that  of  uric  acid. 
Excretion  of  these  substances  is  greatly  increased  by  a 
diet  rich  in  nucleins,  as  sweetbreads  and  liver. 

Uric  acid  exists  in  the  urine  in  the  form  of  urates, 
chiefly  of  sodium  and  potassium,  which  in  concentrated 
urines  are  readily  thrown  out  of  solution  and  constitute 
the  familiar  sediment  of  "amorphous  urates."  This, 
together  with  the  fact  that  uric  acid  is  frequently  de- 
posited as  crystals,  constitutes  its  chief  interest  to  the 
practitioner.  It  is  a  very  common  error  to  consider 
these  deposits  as  evidence  of  excessive  excretion. 

Pathologically,  the  greatest  increase  of  uric  acid 
occurs  in  leukemia,  where  there  is  extensive  destruction 
of  leukocytes,  in  diseases  with  active  destruction  of  the 
liver  and  other  organs  rich  in  nuclei  and  during  ab- 
sorption of  a  pneumonic  exudate.  There  is  generally 
an  increase  during  x-ray  treatment.  Uric  acid  is  de- 
creased before  an  attack  of  gout  and  increased  for 
several  days  after  it,  but  its  etiologic  relation  is  still 
uncertain.     An  increase  is  also  noted  in  acute  fevers. 

Quantitative  Estimation  of  Piu-in  Bodies. — There  is 
no  accurate  method  which  is  simple  enough  for  clinical 
purposes.     Of  clinical  methods,  the  two  given  here  are 


CHEMICAL   EXAMINATION  1 43 

most  satisfactory.  They  are  based  upon  the  same  prin- 
ciple: precipitation  and  removal  of  phosphates,  and 
then  precipitation  of  purin  bodies  with  silver  nitrate 
which  is  strongly  ammoniated  in  order  to  hold  silver 
chlorid  in  solution.  The  amount  of  purin  bodies  is  cal- 
culated from  the  bulk  of  the  silver-purin,  which  in 
Cook's  method  is  thrown  down  by  the  centrifuge,  and 
in  Hall's  is  allowed  to  settle  for  twenty-four  hours. 
The  urine  must  be  albumin-free. 

1.  Cook's  Method. — ^In  a  centrifuge  tube  take  lo  c.c. 
urine  and  add  about  i  Gm.  (about  i  c.c.)  sodium  carbonate 
and  I  or  2  c.c.  strong  ammonia.  Shake  until  the  soda  is 
dissolved.  The  earthy  phosphates  will  be  precipitated. 
Centrifugalize  thoroughly  and  pour  off  all  the  clear  fluid 
into  a  graduated  centrifuge  tube.  To  this  fluid  add  2  c.c. 
ammonia  and  2  c.c.  ammoniated  silver  nitrate  solution. 
Let  stand  a  few  minutes,  and  revolve  in  the  centrifuge  until 
the  bulk  of  precipitate  remains  constant.  Each  o.i  c.c.  of 
sediment  represents  0.001176  Gm.  purin  bodies. 

Ammoniated  silver  nitrate  solution  is  prepared  by  dissolv- 
ing 5  Gm.  of  silver  nitrate  in  100  c.c  distilled  water,  and 
adding  ammonia  until  the  solution  clouds  and  again  be- 
comes clear. 

2,  Hall's  Method. — The  instrument  is  shown  in  Fig.  39. 
Close  the  stop-cock,  introduce  90  c.c.  urine  and  20  c.c.  of 
the  magnesia  solution,  and  mix  by  inverting  a  few  times. 
Open  the  stop-cock  and  let  the  instrument  stand  for  about 
ten  minutes,  or  until  the  precipitated  phosphates  have  set- 
tled into  the  lower  chamber.  Then  close  the  stop-cock, 
and  pour  in  ammoniated  silver  nitrate  solution  until  the 
level  of  the  fluid  reaches  the  loo-c.c.  mark.  Mix  well, 
and  if  any  white  precipitate  of  silver  chlorid  persists,  bring 
it  into  solution  by  adding  a  few  drops  of  ammonia.     Stand 


144 


THE   URINE 


u 

tern 
M 


E90 


E60 


the  instrument  in  the  dark  for  twenty-four  hours  and 
read  off  the  bulk  of  the  precipitate.  The  corresponding 
percentage  of  purin  nitrogen  is  found  by  reference  to  a 
table  which  accompanies  the  instru- 
ment. Albumin  must  be  removed  be- 
fore making  the  test. 

The  magnesia  mixture  is  prepared  by 
dissolving  lo  Gm.  of  magnesium  chlorid 
in  75  c.c.  of  water  and  adding  lo  Gm.  of 
ammonium  chlorid  and  loo  c.c.  strong 
ammonium  hydroxid.  If  a  precipitate 
forms,  it  is  dissolved  by  further  addition 
of  ammonia.  Add  water  to  bring  the 
volume  to  200  c.c.  and  finally  add  10 
Gm.  of  finely  powdered  talcum. 

The  ammoniated  silver  nitrate  solution 
used  in  Hall's  method  consists  of  silver 
nitrate,  i  Gm.;  ammonium  hydroxid, 
100  c.c;  talcum,  5  Gm.;  distilled  water, 
100  c.c. 

■Quantitative  Estimation  of  Uric 

Acid.— Ruhemann's  method,  while 
far  from  accurate,  will  probably 
answer  for  clinical  work.  The  esti- 
mation is,  however,  seldom  of  any 
clinical  value. 


:50 


E5I) 


Fig.  39. — Hall's 
purinometer. 


Ruhemann's  Method  for  Uric  Acid. — The  urine  must  be 
slightly  acid.  By  means  of  a  pipet  fill  Ruhemann's  tube 
(Fig.  40)  to  the  mark  5  with  the  indicator,  carbon  disul- 
phid,  so  that  the  lowest  part  of  the  meniscus  is  on  a  level 
with  the  mark,  as  indicated  in  Fig.  40.  Next  add  Ruhe- 
mann's reagent  until  the  base  of  the  upper  arch  of  the 
meniscus  is  level  with  the  mark  /.     The  carbon  disulphid 


CHEMICAL   EXAMINATION 


145 


will  assume  a  violet  color.  Add  the  urine,  a 
small  quantity  at  a  time,  closing  the  tube 
with  the  glass  stopper  and  shaking  vigor- 
ously after  each  addition,  until  the  disulphid 
loses  every  trace  of  its  violet  color  and  be- 
comes pure  white.  This  completes  the  test. 
Toward  the  end  the  reagent  should  be 
added  a  very  little  at  a  time,  and  the 
shaking  should  be  prolonged  in  order  not 
to  pass  the  end-point.  The  figure  in  the 
right-hand  column  of  figures  corresponding 
to  the  top  of  the  fluid  gives  the  amount  of 
uric  acid  in  parts  per  thousand.  The 
presence  of  diacetic  acid  interferes  with  the 
test,  as  do  also,  to  some  extent,  bile  and 
albumin.  Diacetic  acid  can  be  driven  oflf 
by  boiling;  bile-pigment  and  albumin  are 
removed  as  described  elsewhere  (see  pp. 
loi  and  166). 

Ruhemann's  reagent  consists  of  iodin,  0.5 
Gm.;  potassium  iodid,  1.25  Gm.;  absolute 
alcohol,  7.5  Gm.;  glycerol,  5  Gm.;  distilled 
water  to  100  c.c. 

6.  Ammonia. — A  small  amount  of 
ammonia,  combined  with  hydrochloric, 
phosphoric,  and  sulphuric  acids,  is  always 
present.  Estimated  as  NH3,  the  normal 
average  is  about  0.7  Gm.  in  twenty-four 
hours.  This  represents  4  to  5  per  cent, 
of  the  total  nitrogen  of  the  urine,  ammonia 
standing  next  to  urea  in  this  respect. 

Under  ordinary  conditions,  most  of 
the  ammonia  which  results  from  the 
10 


?    e!8 


i.i 


^i.il-l.89 

'"'   3.15 


146  THE    URINE 

metabolic  processes  is  transformed  into  urea.  When, 
however,  acids  are  present  in  excess,  either  from  inges- 
tion of  mineral  acids  or  from  abnormal  production  of 
acids  within  the  body  (as  in  fevers,  diabetes,  pernicious 
vomiting  of  pregnancy,  delayed  chloroform-poisoning, 
etc.),  ammonia  combines  with  them  and  is  so  excreted, 
urea  being  correspondingly  decreased.  It  is  thus  that 
the  body  protects  itself  against  acid  intoxication.  A 
marked  increase  of  ammonia  is,  therefore,  very  im- 
portant as  an  index  of  the  tendency  to  acidosis,  par- 
ticularly that  associated  with  the  presence  of  diacetic 
and  oxybutyric  acids. 

In  diabetes  mellitus  ammonia  elimination  may  reach 
4  or  5  Gm.  daily.  It  is  likewise  markedly  increased  in 
pernicious  vomiting  of  pregnancy,  but  not  in  nervous 
vomiting;  and  in  conditions  in  which  the  power  to  syn- 
thesize urea  is  interfered  with,  notably  cirrhosis  and 
other  destructive  diseases  of  the  liver  and  conditions 
associated  with  deficient  oxygenation.  Certain  drugs 
have  a  marked  influence  upon  ammonia  elimination; 
thus,  fixed  alkalies  and  salts  of  organic  acids  diminish 
it,  while  inorganic  acids  such  as  hydrochloric  increase  it. 

Quantitative  Estimation. — The  urine  must  be  fresh, 
since  decomposition  increases  the  ammonia.  The. 
formalin  method  is  satisfactory  for  clinical  work  though 
subject  to  some  inaccuracies.  When  carried  out  with- 
out use  of  lead  acetate,  it  includes  amino-acids  with  the 
ammonia,  hence  gives  figures  that  are  too  high.  The 
Folin  and  Denis  method  gives  ammonia  only  and  is 
accurate.  The  difference  between  the  figures  obtained 
by  the  two  methods  therefore  represents  amino-acids. 


CHEMICAL   EXAMINATION  I47 

Ronchese-Malfatti  Formalin  Method. — This  depends 
upon  the  fact  that  when  formahn  is  added  to  the  urine  the 
ammonia  combines  with  it,  forming  hexamethylenamin. 
The  acids  with  which  the  ammonia  was  combined  are  set 
free,  and  their  quantity,  ascertained  by  titration  with  sodium 
hydroxid,  indicates  the  amount  of  ammonia. 

Take  10  c.c.  of  the  urine  in  a  beaker  or  evaporating  dish, 
add  50  c.c.  water  and  10  drops  of  0.5  per  cent,  alcohohc  solu- 
tion of  phenolphthalein.  Neutralize  by  adding  a  weak 
caustic  soda  or  sodium  carbonate  solution  until  a  permanent 
pink  color  appears.  To  5  c.c.  formalin  add  15  c.c.  water 
and  neutralize  in  the  same  way.  Pour  the  formalin  into  the 
urine.  The  pink  color  at  once  disappears,  owing  to  libera- 
tion of  acids.  Now  add  decinormal  sodium  hydroxid 
solution  from  a  buret  until  the  pink  color  just  returns. 
Each  cubic  centimeter  of  the  decinormal  solution  used  in 
this  titration  corresponds  to  0.0017  Gm.  of  NH3.  This  must 
be  multiplied  by  10  to  obtain  the  percentage  from  which  the 
twenty-four-hour  elimination  of  ammonia  is  calculated. 

The  method  is  more  complicated,  but  distinctly  more 
accurate,  when  carried  out  as  suggested  by  E.  W.  Brown: 
Treat  60  c.c.  of  urine  with  3  Gm.  of  basic  lead  acetate,  stir 
well,  let  stand  a  few  minutes,  and  filter.  This  removes  cer- 
tain interfering  nitrogenous  substances.  Treat  the  filtrate 
with  2  Gm.  neutral  potassium  oxalate,  stir  well,  and  filter. 
Take  10  c.c.  of  the  filtrate,  add  50  c.c.  water  and  15  Gm. 
neutral  potassium  oxalate,  and  proceed  with  the  ammonia 
estimation  as  above  outlined. 

Method  of  Folin  and  Denis. — The  reagents  used  are  the 
same  as  those  already  given  for  the  similar  urea  method. 
(See  page  140.) 

I.  To  10  c.c.  of  urine  in  a  small  flask  add  i  c.c.  of  25  per 
cent,  meta-phosphoric  acid,  9  c.c.  distilled  water,  and 
2  Gm.  Merck's  blood  charcoal.  Shake  for  at  least  one 
minute  and  filter. 


148  THE    URINE 

2.  Transfer  2  c.c.  of  the  filtrate  to  a  loo-c.c.  flask,  add 
about  70  c.c.  distilled  water  and  15  c.c.  Nessler's  solution, 
make  up  to  100  c.c.  with  distilled  water  and  mix  well. 

3.  Make  up  a  standard  solution  consisting  of  20  c.c. 
standard  ammonium  sulphate  solution  (representing  o.ooi 
Gm.  ammonia  nitrogen),  15  c.c.  Nessler's  solution  and 
water  to  make  exactly  100  c.c. 

4.  Find  the  amount  of  nitrogen  in  the  unknown  solution 
by  comparing  it  with  the  standard  in  a  colorimeter.  This 
amount  then  represents  the  ammonia  nitrogen  in  i  c.c.  of 
urine.  Multiplied  by  100,  it  gives  the  percentage  of 
ammonia  nitrogen.  To  transform  these  figures  into  terms 
of  NH3  multiply  by  1.2 14. 

7.  Amylase. — A  small  quantity  of  starch-digesting 
ferment — derived  chiefly  from  the  pancreas — can  be 
detected  in  the  urine  of  healthy  persons.  According 
to  Brown  under  normal  conditions  the  twenty-four- 
hour  urine  will  digest  1500  to  12,000  c.c.  of  i  per  cent, 
starch  solution  in  one-half  hour  at  38°C.;  the  normal 
amount  of  amylase  is  therefore  said  to  be  1500  to 
12,000  units.     It  is  somewhat  influenced  by  the  diet. 

Amylase  is  diminished  in  pancreatic  disease  and  in 
nephritis  with  deficient  renal  permeaDility.  It  is  in- 
creased in  simple  obstruction  of  the  pancreatic  duct, 
although  as  the  pancreas  becomes  involved  in  the  path- 
ologic process  the  amount  diminishes.  The  estimation 
of  urinary  amylase  is  therefore  important  in  suspected 
disease  of  the  pancreas,  particularly  when  considered 
in  connection  with  the  pancreatic  ferments  of  the  feces. 
It  has  also  been  proposed  as  a  test  of  renal  function 
but  does  not  promise  much  in  this  field. 

Estimation  of  Amylase. — i.  Obtain  the  twenty-four- 
hour  urine,  which  must  be  kept  in  a  cool  place  and  may  be 


CHEAnCAL   EXAMINATION  I49 

preserved  by  addition  of  an  ounce  of  toluol.     It  should  be 
examined  without  delay. 

2.  Dilute  the  urine  to  3000  c.c.  and  mix  well. 

3.  Proceed  exactly  as  for  fecal  amylase,  steps  i  to 
5,  except  that  a  o.i  per  cent,  starch  solution  must  be 
substituted  for  the  i  per  cent,  solution  recommended  for 
the  feces,  and  a  weaker  iodin  solution  must  be  used. 
One  part  of  Gram's  iodin  solution  diluted  with  4  parts  of 
water  will  answer. 

The  normal  falls  between  tube  8  (1500  units)  and  tube 
II  (12000  units). 

B.  Abnormal  Constituents 

Those  substances  which  appear  in  the  urine  only  in 
pathologic  conditions  are  of  much  more  interest  to  the 
clinician  than  are  those  which  have  just  been  discussed. 
Among  them  are:  proteins,  sugars,  the  acetone  bodies, 
bile,  urobilin,  hemoglobin,  hematoporphyrin  and  the 
diazo  substances.  The  detection  of  drugs  in  the  urine 
will  also  be  discussed  under  this  head. 

1,  Proteins. — Of  the  proteins  which  may  appear  in 
the  urine,  serum-albumin  and  serum-globulin  are  the 
most  important.  Mucin,  proteose,  and  a  few  others  are 
found  occasionally,  but  are  of  less  interest. 

( I )  Serum-albmnin  and  Senim-globiilin. — These  two 
proteins  constitute  the  so-called  "urinary  albumin." 
They  usually  occur  together,  have  practically  the  same 
significance,  and  both  respond  to  all  the  ordinary  tests 
for  "albumin." 

Their  presence,  or  albuminuria,  is  probably  the  most 
important  pathologic  condition  of  the  urine.  It  is 
either  accidental  or  renal.     The  physician  can  make  no 


150  THE    URINE 

greater  mistake  than  to  regard  all  cases  of  albuminuria 
as  indicating  kidney  disease. 

Accidental  or  false  albuminuria  is  due  to  admixture 
with  the  urine  of  albuminous  fluids,  such  as  pus,  blood, 
and  vaginal  discharge.  The  microscope  will  usually 
reveal  its  nature.  It  occurs  most  frequently  in  pyelitis, 
cystitis,  and  chronic  vaginitis,  and  the  quantity  is  usu- 
ally small. 

Renal  albuminuria  refers  to  albumin  which  has  passed 
from  the  blood  into  the  urine  through  the  walls  of  the 
kidney  tubules  or  the  glomeruli. 

Albuminuria  sufficient  to  be  recognized  by  clinical 
methods  probably  never  occurs  as  a  physiologic  condi- 
tion, the  so-called  physiologic  albuminuria  appearing 
only  under  conditions  which  must  be  regarded  as  ab- 
normal. Among  these  may  be  mentioned  excessive 
muscular  exertion  in  those  unaccustomed  to  it;  exces- 
sive ingestion  of  proteins;  prolonged  cold  baths;  and 
childbirth.  In  these  conditions  the  albuminuria  is 
slight  and  transient. 

There  are  certain  other  forms  of  albuminu^-ia  which 
have  still  less  claim  to  be  called  physiologic,  but  which 
are  not  always  regarded  as  pathologic.  Among  these 
are  cyclic  albuminuria,  which  regularly  recurs  at  a  cer- 
tain period  of  the  day,  and  orthostatic  ox  postural  albiunin- 
uria,  which  appears  only  when  the  patient  is  standing. 
They  are  rare  and  of  obscure  origin,  and  occur  for  the 
most  part  in  neurasthenic  subjects  during  adolescence. 
It  is  noteworthy  in  this  connection  that  nephritis  some- 
times begins  with  a  cyclic  albuminuria. 

In  pathologic  conditions  and  in  most,  at  least,  of  the 
"functional"    conditions    just    enumerated,    renal    al- 


CHEMICAL   EXAMINATION  151 

buminuria  may  be  referred  to  one  or  more  of  the  follow- 
ing causes.  In  nearly  all  cases  it  is  accompanied  by 
tube-casts. 

(a)  Changes  in  the  blood  which  render  its  albumin 
more  diffusible,  as  in  severe  anemias,  purpura,  and 
scurvy.     Here  the  albumin  is  small  in  amount. 

(b)  Changes  in  circulation  in  the  kidney,  either  anemia 
or  congestion,  as  in  excessive  exercise,  chronic  heart 
disease,  and  pressure  upon  the  renal  veins.  The  quan- 
tity of  albumin  is  usually,  but  not  always,  small.  Its 
presence  is  constant  or  temporary,  according  to  the 
cause.  Most  of  the  causes,  if  continued,  will  produce 
organic'Changes  in  the  kidney. 

(c)  Orgastic  Changes  in  the  Kidney. — These  include 
the  inflammatory  and  degenerative  changes  commonly 
grouped  together  under  the  name  of  nephritis,  and  also 
renal  tuberculosis,  neoplasms,  and  cloudy  swelling  due 
to  irritation  of  toxins  and  drugs.  The  amount  of  al- 
bumin eliminated  in  these  conditions  varies  from  minute 
traces  to  20  Gm.,  or  even  more,  in  the  twenty-four  hours, 
and,  except  in  acute  processes,  bears  little  relation  to 
the  severity  of  the  disease.  In  acute  and  chronic  paren- 
chymatous nephritis  the  quantity  is  usually  very  large. 
In  chronic  interstitial  nephritis  it  is  small — frequently 
no  more  than  a  trace.  It  is  small  in  cloudy  swelling 
from  toxins  and  drugs,  and  variable  in  renal  tuber- 
culosis and  neoplasms.  In  amyloid  disease  of  the  kid- 
nvjy  the  quantity  is  usually  small,  and  serum-globulin 
may  be  present  in  especially  large  proportion,  or  even 
alone.  Roughly  distinctive  of  serum-globulin  is  the 
appearance  of  an'  opalescent  cloud  when  a  few  drops  of 
the  urine  are  dropped  into  a  glass  of  distilled  water. 


152  THE    URINE 

Detection  of  albumin  depends  upon  its  precipitation 
by  chemicals  or  coagulation  by  heat.  There  are  many 
tests,  but  none  is  entirely  satisfactory,  because  other 
substances  as  well  as  albumin  are  precipitated.  The 
most  common  source  of  error  is  mucin.  When  any 
considerable  amount  of  mucin  is  present  it  can  be 
removed  by  acidifying  with  acetic  acid  and  filtering. 
Urine  voided  early  in  the  evening  or  a  few  hours  after 
a  meal  is  most  likely  to  contain  albumin. 

It  is  very  important  that  urine  to  be  tested  for  albumin 
be  rendered  clear  by  filtration  or  centrifugation.  This 
is  too  often  neglected  in  routine  work.  When  ordinary 
methods  do  not  sufi&ce,  it  can  usually  be  cleared  by 
shaking  up  with  a  little  purified  talc,  infusorial  earth  or 
animal  charcoal  and  filtering.  This  will  remove  a  part 
of  the  albumin  by  adsorption,  but  the  remainder  is 
more  easily  detected.  If  the  urine  is  alkaline,  sufficient 
acetic  acid  should  be  added  to  make  it  acid  to  litmus. 
Vaughan  has  recently  called  attention  to  the  fact  that 
if  bacteria  be  abundant  in  an  alkaline  urine,  some  of  the 
bacterial  proteins  may  go  into  solution  and  give  the 
tests  for  albumin. 

Technic  of  Ring  or  Contact  Tests. — Since  this  simple 
and  widely  useful  method  of  testing  is  best  known  in  con- 
nection with  the  detection  of  albumin  a  general  description  is 
given  at  this  place. 

Take  a  few  cubic  centimeters  of  the  heavier  fluid  in  a 
conical  test-glass,  hold  the  glass  in  an  inclined  position, 
and  run  the  lighter  fluid  gently  down  the  inside  of  the 
glass  by  means  of  a  medicine-dropper  so  that  it  will  form 
a  layer  on  top  of  the  other  without  mixing.  In  the  case  of 
the  urine,  which  must  be  filtered  before  testing,  it  may  be 
run  in  directly  from  the  stem  of  the  funnel  by  touching 


CHEMICAL   EXAMINATION  1 53 

this  against  the  wall  of  the  test  glass.  If  the  test  be  posi- 
tive a  sharply  defined  white  or  colored  ring  will  appear 
where  the  two  fluids  come  into  contact.  According  to  its 
color  the  ring  is  seen  most  clearly  if  viewed  against  a  white 
or  a  black  background,  as  the  case  may  be;  and  one  side 
of  the  test-glass  may  be  painted  half  white,  half  black, 
for  this  purpose. 

In  the  writer's  experience  this  is  the  most  satisfactory 
technic.  The  common  practice  of  taking  the  reagent  in  a 
narrow  test-tube  and  pouring  the  urine  in  on  top  of  it 
from  a  bottle  is  much  inferior.  Boston  brings  the  fluids 
into  contact  in  a  glass  pipet  which  is  immersed  first  in 
the  lighter  fluid  and  then  (after  wiping  the  outside  of  the 
pipet)  in  the  heavier.  This  is  convenient  for  the  routine 
testing  of  a  large  number  of  urines,  but  cannot  be  recom- 
mended for  accuracy,  owing  to  the  small  diameter  of  the 
column  of  fluid.  Substitution  of  a  medicine-dropper  in 
place  of  the  pipet  renders  Boston's  method  more  convenient 
but  no  more  accurate.  For  those  who  do  only  a  little 
testing  the  "  horismascope "  (Fig.  41)  will  be  found  very 
convenient  and  satisfactory.  The  instrument  is,  however, 
fragile  and  somewhat  expensive. 

The  albumin  tests  here  given  are  widely  used  and  can 
be  recornmended  for  clinical  purposes.  They  make  no 
distinction  between  serum-albumin  and  serum-globulin. 
As  a  rule  the  most  sensitive  tests  are  not  the  most 
useful  for  clinical  purposes.  The  writer  prefers 
Purdy's  heat  test  and  Roberts'  ring  test  for  routine 
testing,  but  other  workers  will  have  other  favorites. 
The  extremely  sensitive  trichloracetic  and  sulphosali- 
cylic  acid  tests  are  used  only  in  special  cases. 

I.  Trichloracetic  Acid  Test. — The  reagent  consists  of  a 
saturated  aqueous  solution  of  trichloracetic  acid  to  which 


154  THE    URINE 

magnesium  sulphate  is  added  to  saturation.  A  simple 
saturated  solution  of  the  acid  may  be  used,  but  addition  of 
magnesium  sulphate  favors  precipitation  of  globulin,  and, 
by  raising  the  specific  gravity,  makes  the  test  easier  to 
apply. 

The  test  is  carried  out  by  the  "ring"  or  "contact" 
method  just  described.  If  albumin  be  present,  a  white, 
cloudy  ring  will  appear  where  the  two  fluids  come  in 
contact. 

This  is  an  extremely  sensitive  test,  but,  unfortunately, 
both  mucin  and  proteoses  respond  to  it;  urates,  when  abun- 
dant, may  give  a  confusing  white  ring,  and  the  reagent  is 
comparatively  expensive.  It  is  not  much  used  in  routine 
work  except  as  a  control  to  the  less  sensitive  tests. 

2.  Stilphosalicylic  acid  in  20  per  cent,  aqueous  solution 
may  be  used  in  the  same  way  as  the  trichloracetic  acid 
reagent.  It  is  fully  as  sensitive  and  is  somewhat  more 
reliable  in  that  urates  and  resins  are  not  precipitated. 
This  may  also  be  applied  by  adding  a  few  drops  of  the 
reagent  to  a  few  cubic  centimeters  of  the  urine  in  a  test- 
tube  and  obtaining  a  white  cloud  in  the  presence  of  albumin, 
or  by  adding  a  bit  of  the  sulphosalicylic  acid  in  the  solid 
state.  The  last  is  especially  convenient  for  the  practi- 
tioner who  wishes  to  make  albumin  tests  at  the  bedside. 

3.  Roberts'  Test. — The  reagent  consists  of  pure  nitric 
acid,  I  part,  and  saturated  aqueous  solution  of  magnesium 
sulphate,  5  parts.  It  is  applied  by  the  "ring"  or  "con- 
tact" method  above  described. 

Albumin  gives  a  white  ring,  which  varies  in  density  with 
the  amount  present  and  when  traces  only  are  present,  may 
not  appear  for  two  or  three  minutes.  A  similar  white  ring 
may  be  produced  by  primary  proteose,  thymol,  and  resin- 
ous drugs.  White  rings  or  cloudiness  in  the  urine  above 
the  zone  of  contact  may  result  from  excess  of  urates  or 
mucus.     Colored    rings    near    the    junction    of    the    fluids 


CHEMICAL   EXAMINATION  1 55 

may  be  produced  by  iodids,  urinary  pigments,  bile,  or 
indican,  but  these  are  not  so  frequent  as  with  Heller's 
test. 

Roberts'  test  is  one  of  the  best  for  routine  work,  although 
the  various  rings  are  apt  to  be  confusing  to  the  inex- 
perienced.    It  is  more  sensitive  than  Heller's  test,  of  which 


Fig.  41. — Horismascope:  adding  the  reagent. 

it  is  a  modification,  and  has  the  additional  advantage  that 
the  reagent  is  not  so  corrosive. 

4.  Ulrich's  test  avoids  the  somewhat  confusing  colored 
rings.  The  reagent  consists  of  saturated  solution  of 
common  salt,  98  c.c. ;  glacial  acetic  acid,  2  c.c.  It  must  be 
perfectly  clear.  Boil  a  few  cubic  centimeters  of  this  fluid 
in  a  test-tube,  and  immediately  overlay  with  the  urine  as 
in  the  preceding  tests.  Albumin  and  globulin  give  a  white 
ring  at  the  zone  of  contact. 


156  THE  rmxE 

5.  Ihadf^f*  Heat  Test— Take  a  test-tube  two-thirds  full 
of  ■dnty  add  aboat  OQe-axth  its  volume  of  saturated  scAu- 
tiaa  of  soA^B  cMackl,  and  5  to  10  drc^  of  50  per  cent. 
acetic  add.  Mix,  and  b<xl  the  upper  inch,  holding  the 
tube  with  the  faagexs  near  the  bottom.  A  white  cloud 
m  the  heated  partka  ^cms  the  presence  ci  albumin.  A 
(bat  doad  is  best  seea  when  viewed  against  a  black 
backgnxmd  at  a  Mct^nt-^  of  two  or  three  feet. 

This  is  a  valuafafe  test  ior  routine  work.  It  is  simple, 
sufficiently  accurate  for  dinical  pmposes,  and  has  prac- 
tically DO  fafl**-««»g  Addiii^m  ff  tie  saU  stiwH&m,  by  raising 
the  spec^  ffwutj,  praemts  fndpH^iiom  tf  wukul.  Bence- 
JoDes'  protein  may  produce  a  white  doud,  which  disap- 
pears upon  boffii^  and  reappears  upon  cooling. 

6.  Heat  wad.  Wtnc  Add  Test— This  is  one  of  the  ddest 
of  the  albumin  tests,  and,  if  propoly  carried  out,  one  of  the 
best.  Boil  about  5  cc  of  fflteied  urine  in  a  test-tube  and 
add  I  to  3  drops  of  concentiated  nitric  add.  The  tube 
may  be  held  with  a  test-tube  damp  or  simply  with  a  strip 
cf  muslin,  the  center  of  which  is  folded  once  aroimd  the 
neck  of  the  tube.  A  white  dood  or  flocailent  predpitate 
(which  nsoaUy  appears  during  the  bcnling,  but  if  the  qu^i- 
tity  be  very  small  only  after  adtfition  of  the  add )  denotes 
tihe  pcesence  of  albumin.  A  gwibr  white  predpitate, 
whidi  disappears  up<xi  addition  of  the  add,  is  due  to 
carti^  pbo^ihates.  The  acid  should  not  be  added  before 
htwEu^  and  die  proper  amoant  should  always  be  used; 
odterwise,  part  of  the  albumin  may  fail  to  be  precipitated 
or  may  be  trans&)rmed  to  add-albimiin  and  redissolved. 

A  dedded  advantage  of  this  test  is  the  fact  that  it  allows 
a  loo^  estimation  of  the  amount  of  albumin  frcm  the 
tw^nuii.  of  the  wtCmJ-n*  after  standing  over  ni^t.  When 
tihe  entire  ftdd  miImIjCih.  the  albumin  amounts  to  2  to  3 
per  coit.  Sediments  reaching  to  one-half,  one-third,  ooe- 
foorth  and  one-t«ith  the  hd^t  of  the  column  oi  urine 


rwFinriif.  wxAMisAnom  157 

conespond  nspedivchr  to  aboot  i,  aL5,  OL25,  aad  oli. 
per  cenL  albaiinii.    Wben  diere  b  aaiy  a  digptt 
die  aDmran  does  not  cxcsed  ouoi  per  cent. 

QmntiiififC  BiJiiiiiiliiw — Tlie  giavimetxi^ 
the  most  r^afale  metliod,  is  too  dabcaate  for 
woA.    The  three  vIdcIi  foflav  aic  sinple 
and  are  \ay  viddy^  Bsed  but  noBC  is  cb- 
tirdy  sati^iactorf. 

I.  EdacVlB  McliMid.— The  vine  aoBt  be 
dear,  of  add  icactina,  and  not  conccntiaied- 
Ahrajs  filter  bdore  trst^g,  aad,  H  mm'^jiji, 
add  acetic  add  and  dBote  «i&  vatcr,  nnknig 
allowance  f cr  the  dHntiua  in  the  find  calcn- 
latian.    Esbach^  tube  (F%.  43)  is  < 
a  test-tobe  vith  a  naik  U  near  the 
a  maik  R  near  the  top,  and  giadnatinns        llll5 
K,  I,  2,  3,  etc,  near  the  botftoa.    FB  ttr         |f ' 
tnbe  to  the  nmk  U  vith  mme  and  to  thr  -f-^ 

maik  R  vith  the  reagent.    Clase  viih  a  nd>-  ^ 

her  stopper,  invert  slonlf  sevenl  tscs^  and  \^ 

set  aside  in  a  cod  piirr  At  the  end  of 
twcntf-foar  hons  read  off  the  heq^  ot 
the  pndqpitate.  TUs  gpves  the  j—nl  of 
»B— iBwn  Hk  cvn^n  fcr  i^br  m^J  ^kmsM  ^t 
ihiiei  ij  10  i»  Maim  He  ptnewtmgt. 

heak.  adYises  mhRtiun  of  a  saal  q—ntifj  of 
chaimal,  pnnioc  or  knofin  after  addfag  E^inch's 
Tbis   hastms  sefnentation  viidi   is  complete 
minnlffs  to  half  an  boor.    Andreses  nses  oli  to  ol.2  Gb. 
of  baiiom  sn^ihatc 

EAmcfs  rtrngad  Misisls  of  picnc  add,  i  Gln^  dtnc 
add.  2  (dL,  and  dbtScd  vater,  to  mnke  100  ex. 

3.  TSnchiya^  Mf,ftod — This  is  carried  ont  in  the 
■anner  as  the  Esbnch  sKthod,  nsine  the  faDopn^ 


158  THE    URINE 

Phosphotungstic  acid 1.5  Gm.; 

Alcohol  (96  per  cent.) 95.00.0.; 

Concentrated  hydrochloric  acid 5.0  c.c. 

The  urine  should  be  diluted  to  a  specific  gravity  not  exceed- 
ing 1.008.  The  method  is  said  to  be  much  more  accurate 
than  the  original  Esbach  method,  particularly  with  small 
quantities  of  albumin,  but  in  the  writer's  work  this  has 
not  proved  to  be  true. 

3.  Purdy's  Centrifugal  Method. — This  is  detailed  in 
the  table  on  opposite  page.  Since  10  c.c.  of  urine  were  used, 
each  0.1  c.c.  of  precipitate  is  i  per  cent,  by  bulk. 

(2)  Mucin. — Traces  of  the  substances  (mucin,  mu- 
coid, nucleo-protein,  etc.)  which  are  loosely  classed 
under  this  name  are  present  in  normal  urine;  increased 
amounts  are  observed  in  irritations  and  inflammations 
of  the  mucous  membrane  of  the  urinary  tract.  They 
are  of  interest  chiefly  because  they  may  be  mistaken 
for  albumin  in  most  of  the  tests.  If  the  urine  be  diluted 
with  water  and  acidified  with  acetic  acid,  the  appearance 
of  a  white  cloud  indicates  the  presence  of  mucin. 

Mucin  and  mucoid  are  glyco-proteins,  and  upon  boil- 
ing with  an  acid  or  alkali,  as  in  Fehling's  test,  yield  a 
carbohydrate  substance  which  reduces  copper. 

(3)  Bence-Jones'  Protein.— The  protein  known  by 
this  name  was  originally  classed  as  an  albumose,  but  its 
protein  nature  is  now  well  established.  It  was  formerly 
regarded  as  practically  pathognomonic  of  multiple 
myeloma  but  has  recently  been  found  in  a  number  of 
cases  of  chronic  leukemia,  of  both  lymphatic  and 
myelogenous  types  and  in  osteomalacia. 

To  detect  Bence-Jones'  protein  the  urine  is  slightly 
acidified  with  acetic  acid  and  gently  heated  in  a  water-bath. 


CHEMICAL   EXAMINATION 


159 


PURDY'S  QUANTITATIVE  METHOD  FOR  ALBUMIN  IN 
URINE  (CENTRIFUGAL) 

Table  showing  the  relation  between  the  volumetric  and  gravimetric  percentage  oj 
albumin  obtained  by  means  of  the  centrifuge  with  radius  of  six  and  three-quarter 
inches;  rate  of  speed,  1500  revolutions  per  minute;  time,  three  minutes. 


Volumetric 
percentage 
by  centri- 
fuge. 

Percentage 
by  weight 

of  dry 
albumin. 

Volumetric 
percentage 
by  centri- 
fuge. 

Percentage 

by  weight 

of  dry 

albumin. 

Volumetric 
percentage 
by  centri- 
fuge. 

Percentage 
by  weight 

of  dry 
albumin. 

>4 

0.003 

133-2 

0.281 

3i>^ 

0.656 

Vt 

0 .01          : 

14 

0.292 

32 

0.667 

H 

0.016       1 

I4>2 

0.302 

32H 

0.677 

I 

0.021 

IS 

0.313 

33 

0.687 

i>i 

0.026 

I5>i 

0.323 

33H 

0.698 

iH 

0.031 

16 

0.333 

34 

0.708 

iH 

0.036 

16H 

0.344 

34}-! 

0.719 

2 

0.042 

17 

0.3S4 

35 

0.729 

2>i 

0.047       , 

I7>2 

0 .  365 

3S>^ 

0.74 

2H 

0.052       1 

18 

0    375 

36 

0.7S 

2?i 

0.057 

I8H 

0.385 

36H 

0.76 

3 

0 .  063 

19 

0.396 

37 

0.771 

3M 

0  .068 

19>^ 

0  .  406 

373^ 

0.781 

3>^ 

0.073        i 

20 

0.417 

38 

0.792 

3?i 

0.078 

20J2 

0.427 

38H 

0.801 

4 

0.083        1 

21 

0   438 

39 

0.813 

4>4 

0.089 

2I>2 

0.448 

39>^ 

0.823 

Ahi 

0 . 094        ' 

22 

0.458 

40 

0.833 

4K 

0.099 

22>;; 

0.469 

40>$ 

0.844 

5 

0    104 

23 

0    479 

41 

0.854 

S>^ 

0  . 1 1 1 

233-2 

0.49 

41H 

0.86s 

6 

0.I2S 

1              24 

0.5 

42 

0.87s 

6H 

0.13s 

:        24J.2 

■     0   51 

42M 

0.88s 

7 

0    146 

!             25 

0.521 

43 

0.896 

7H 

0 .  1.56 

25>2 

0.531 

43H 

0.906 

8 

0.  167 

26 

0.542 

44 

0.917 

sy2 

0.  177 

26>2 

0.552 

44M 

0.927 

9 

0.187 

27 

0.563 

4S 

0.938 

9H 

0. 198 

273^2 

0.573 

4.SH 

0.948 

10 

0  208 

28 

0.583 

46 

0.958 

lOH 

0.219 

28>i 

0.594 

463-^ 

0.069 

II 

0.229 

i       29 

0.604 

47 

0.979 

lo'i 

0.24 

29}2 

0.6lS 

47H 

0.99 

12 

0.2s 

'              30 

0.625 

48 

I  .0 

I2M 

0.26 

30>2 

0   635 

13 

0 .  271 

31 

0   646 

Test. — Three  cubic  centimeters  of  10  per  cent,  solution  of  ferrocyanid  of 
potassium  and  2  cubic  centimeters  of  50  per  cent,  acetic  acid  are  added  to  10 
cubic  centimeters  of  the  urine  in  the  percentage  tube  and  stood  aside  for  ten 
minutes,  then  placed  in  the  centrifuge  and  revolved  at  rate  of  speed  and  time  as 
stated  at  head  of  the  table.  If  albumin  is  excessive,  dilute  the  urine  with  water 
until  volume  of  albumin  falls  below  10  per  cent.  Multiply  result  by  the  number 
of  dilutions  employed  before  using  the  table. 


l6o  THE   UEINE 

If  this  substance  be  present,  the  urine  will  begin  to  be 
turbid  at  about  40°C.  and  a  precipitate  will  form  at  about 
6o°C.  As  the  boiling-^oint  is  reached  the  precipitate 
wholly  or  partially  dissolves.  It  reappears  upon  cooling. 
It  may  easily  be  overlooked  in  the  presence  of  albumin. 

(4)  Proteoses. — These  are  intermediate  products  in 
the  digestion  of  proteins  and  are  frequently,  although 
incorrectly,  called  albumoses.  Two  groups  are  gener- 
ally recognized:  primary  proteoses,  which  are  precipi- 
tated upon  half-saturation  of  their  solutions  with 
ammonium  sulphate ;  and  secondary  proteoses,  which  are 
precipitated  only  upon  complete  saturation. 

The  secondary  proteoses  have  been  observed  in  the 
urine  in  febrile  and  malignant  diseases  and  chronic  sup- 
purations, during  resolution  of  pneumonia,  and  in  many 
other  conditions,  but  their  clinical  significance  is  in- 
definite. In  pregnancy,  albumosuria  may  be  due  to 
absorption  of  amniotic  fluid. 

Primary  proteoses  are  rarely  encountered  in  the 
urine. 

The  proteoses  are  not  coagulable  by  heat,"  but  are  precipi- 
tated by  such  substances  as  trichloracetic  acid,  sulpho- 
salicylic  acid,  and  phosphotungstic  acid.  The  primary 
proteoses  alone  are  precipitated  by  concentrated  nitric  acid. 

Proteoses  may  be  detected  by  acidifying  the  urine  with 
acetic  acid,  boiling,  filtering  while  hot  to  remove  mucin, 
albumin,  and  globulin,  and  testing  the  filtrate  by  the  tri- 
chloracetic acid  test.  As  above  indicated,  the  nitric  acid 
test,  and  half  and  complete  saturation  with  ammonium  sul- 
phate, will  separate  the  two  groups. 

2.  Sugars. — Various  sugars  may  at  times  be  found 
in  the  urine.     Dextrose  is  by  far  the  most  common,  and 


CHEMICAL  EXAMINATION  l6l 

is  the  only  one  of  much  cHnical  importance,  Levulose, 
lactose,  and  some  others  are  occasionally  met. 

(i)  Dextrose  (Glucose). — Traces  of  glucose,  too 
small  to  respond  to  the  ordinary  tests,  are  present  in  the 
urine  in  health.  Its  presence  in  appreciable  amount 
constitutes  "glycosuria"  and  is  almost  uniformly  a 
result  of  hyperglycemia. 

Transitory  glycosuria  is  unimportant,  and  may  occur 
in  many  conditions,  as  after  general  anesthesia  and 
administration  of  certain  drugs,  in  pregnancy,  and 
following  shock  and  head  injuries.  Recently  attention 
has  been  directed  to  glycosuria  following  strong  emo- 
tions (anger,  fear,  anxiety)  due,  according  to  Cannon, 
to  increased  adrenal  secretion.  The  urine  of  a  con- 
siderable percentage  of  a  class  of  students  will  give 
positive  tests  for  sugar  following  a  long  and  hard  exam- 
ination. The  possibility  that  a  trace  of  sugar  found 
in  a  patient's  urine  after  a  physical  examination  may  be 
due  to  his  anxiety  must  be  kept  in  mind.  Glycosuria 
may  also  occur  after  eating  excessive  amounts  of  car- 
bohydrates (alimentary  glycosuria).  The  "assimi- 
lation limit"  varies  with  different  individuals  and  with 
different  conditions  of  exercise.  It  also  depends  upon 
the  kind  of  carbohydrate.  The  normal  for  glucose 
is  about  TOO  to  150  Gm.  When  more  than  this  amount 
is  taken  at  one  time  some  of  it  will  be  excreted  in  the 
urine.     Excretion  lasts  for  a  period  of  four  or  five  hours. 

Persistent  glycosuria  has  been  noted  in  brain  injuries 
involving  the  floor  of  the  fourth  ventricle.  As  a  rule, 
however,  persistent  glycosuria  is  diagnostic  of  diabetes 
mellitus,  of  which  disease  it  is  the  essential  symptom. 
The  amount  of  glucose  eliminated  in  diabetes  is  usually 


1 62  THE    URINE 

considerable,  and  is  sometimes  very  large,  reaching  500 
grams,  or  even  more,  in  twenty-four  hours,  but  it  does 
not  bear  any  uniform  relation  to  the  severity  of  the 
disease.  Glucose  may,  on  the  other  hand,  be  almost  or 
entirely  absent  temporarily  and  in  mild  cases  it  may 
appear  only  at  certain  hours  of  the  day. 

Detection  of  Dextrose. — If  albumin  be  present  in 
more  than  traces,  it  must  be  removed  by  boiling  and 
filtering. 

I.  Haines'  Test. — Take  about  4  c.c.  of  Haines'  solution 
in  a  test-tube,  boil,  examine  carefully  for  a  precipitate,  and, 
if  none  is  present,  add  6  or  8  drops  of  urine.  A  heavy  yellow 
or  red  precipitate,  which  settles  readily  to  the  bottom, 
shows  the  presence  of  sugar.  Neither  precipitation  of 
phosphates,  as  a  light,  flocculent  sediment,  nor  simple 
decolorization  of  the  reagent  should  be  mistaken  for  a 
positive  reaction. 

This  is  one  of  the  best  of  the  copper  tests,  all  of  which 
depend  upon  the  fact  that  in  strongly  alkaline  solutions 
glucose  reduces  cupric  hydrate  to  cuprous  hydrate  (yellow) 
or  cuprous  oxid  (red).  They  are  somewhat  inaccurate, 
because  they  make  no  distinction  between  glucose  and  less 
common  forms  of  sugar;  because  certain  normal  substances, 
when  present  in  excess,  especially  mucin,  uric  acid,  and 
creatinin,  may  reduce  copper,  and  because  many  drugs — 
e.g.,  chloral,  chloroform,  copaiba,  acetanilid,  benzoic  acid, 
morphin,  sulphonal,  salicylates — are  eliminated  as  copper- 
reducing  substances.  To  minimize  these  fallacies  dilute 
the  urine,  if  it  be  concentrated;  do  not  add  more  than  the 
specified  amount  of  urine,  and  do  not  boil  after  the  urine  is 
added.  If  chloroform  has  been  used  as  a  preservative,  it 
should  be  removed  by  boiling  the  urine  before  making  the 
test. 


CHEMICAL   EXAMINATION  1 63 

Haines'  solution  is  prepared  as  follows:  Completely  dis- 
solve 2  Gm.  pure  copper  sulphate  in  16  c.c.  distilled  water, 
and  add  16  c.c.  pure  glycerin;  mix  thoroughly,  and  add  156 
c.c.  liquor  potassse.     The  solution  keeps  well. 

2.  Fehling's  Test. — Two  solutions  are  required — one 
containing  34.64  Gm.  pure  crystalline  copper  sulphate  in 
500  c.c.  distilled  water;  the  other,  173  Gm.  Rochelle  salt  and 
100  Gm.  potassium  hydroxid  in  500  c.c.  distilled  water. 
Mix  equal  parts  of  the  two  solutions  in  a  test-tube,  dilute 
with  3  or  4  volumes  of  water,  and  boil.  Add  the  urine  a 
little  at  a  time,  heating,  but  not  boiling,  between  additions. 
In  the  presence  of  glucose  a  heavy  red  or  yellow  precipitate 
will  appear.  The  quantity  of  urine  should  not  exceed 
that  of  the  reagent.  The  fallacies  mentioned  under 
Haines'  test  apply  equally  to  this. 

3.  Benedict's  Test. — This  new  test  promises  to  displace 
all  other  reduction  tests  for  glucose.  The  reagent  is  said  to 
be  ten  times  as  sensitive  as  Haines'  or  Fehling's,  and  not  to 
be  reduced  by  uric  acid,  creatinin,  chloroform,  or  the  alde- 
hyds.     It  consists  of: 

Copper  sulphate  (pure  crystallized) 17.3  Gm.; 

Sodium  or  potassium  citrate 1730  Gm.; 

Sodium  carbonate  (crystallized) 200.0  Gm.; 

(or  100  Gm.  of  the  anhydrous  salt). 
Distilled  water,  to  make 1 000.0  c.c. 

Dissolve  the  citrate  and  carbonate  in  700  c.c.  of  water, 
with  the  aid  of  heat,  and  filter.  Dissolve  the  copper  in 
100  c.c.  of  water  and  pour  slowly  into  the  first  solution, 
stirring  constantly.  Cool,  and  make  up  to  one  liter. 
The  reagent  keeps  indefinitely.  //  can  not  he  used  for 
quantitative  estimations. 

Take  about  5  c.c.  of  this  reagent  in  a  test-tube,  and  add 
8  or  10  drops  (not  more)  of  the  urine.  Heat  to  vigorous 
boiling,  keep  at  this  temperature  for  one  or  two  minutes, 


164 


THE   URTNE 


and  allow  to  cool  slowly.  In  the  presence  of  glucose  the 
entire  body  of  the  solution  will  be  filled  with  a  precipitate, 
which  may  be  red,  yellow,  or  green  in  color.  When  traces 
only  of  glucose  are  present,  the  precipitate  may  appear  only 
upon  cooling.  In  the  absence  of  glucose,  the  solution  re- 
mains clear  or  shows  only  a  faint,  bluish  precipitate,  due  to 
urates. 

4.  Phenylhydrazin  Test. — Kowarsky^s  Method. — ^The  fol- 
lowing directions  include  certain  modifications  which  have 


Fig.  43. — Crystals  of  phenylglucosazone  from  diabetic  urine — Kowar- 
sky's  test  (  X  500). 


been  worked  out  by  C.  S.  Bluemel  in  the  writer's  labora- 
tory: In  a  wide  test-tube  take  5  drops  pure  phenyl- 
hydrazin, 10  drops  glacial  acetic  acid,  and  i  c.c.  saturated 
solution  of  sodium  chlorid.  A  curdy  mass  results.  Add  3 
or  4  c.c.  of  the  urine  and  4  or  5  c.c.  of  water.  Boil  vigor- 
ously for  two  or  three  minutes.  The  annoying  bumping 
can  be  reduced  or  obviated  by  shaking  continually,  or, 
much  better,  by  placing  in  the  test-tube  a  number  of 
pieces  of  glass  tubing,  varying  in  length  from  i}-2   to  3 


CHEMICAL   EXAMINATION  1 65 

inches,  so  as  to  produce  an  organ-pipe  effect.  The  volume 
of  fluid  remaining  after  boiling  should  be  2  to  3  c.c.  Set 
aside  to  cool,  or  if  the  glass  tubes  were  used  pour  the  fluid 
into  another  hot  test-tube  and  allow  to  cool.  Examine  the 
sediment  with  the  microscope,  using  a  two- thirds  objective. 
If  glucose  be  present,  characteristic  crystals  of  phenyl- 
glucosazone  will  be  seen.  These  are  yellow,  needle-like 
crystals  arranged  mostly  in  clusters  or  in  sheaves  (Fig. 
43).  When  traces  only  of  glucose  are  present,  the  crystals 
may  not  appear  for  one-half  hour  or  more.  The  best 
crystals  are  obtained  when  the  fluid  is  cooled  very  slowly. 
It  must  not  be  agitated  during  cooling.  The  test-tubes 
and  pieces  of  tubing  can  be  cleaned  when  necessary  by 
boiling  in  a  solution  of  caustic  soda  or  acetic  acid. 

This  is  an  excellent  test  for  clinical  work.  Bluemel  finds 
that  when  applied  as  above  directed,  with  the  tubing  to  pre- 
vent bumping,  it  will  readily  detect  0.025  P^r  cent,  of 
glucose  in  urine,  the  crystals  appearing  in  three  to  four 
hours.  The  test  has  practically  no  fallacies  excepting 
levulose,  which  is  a  fallacy  for  all  the  ordinary  tests.  Other 
carbohydrates  which  are  capable  of  forming  crystals 
with  phenylhydrazin  are  extremely  unlikely  to  do  so  when 
the  test  is  applied  directly  to  the  urine.  Even  if  not  used 
routinely,  this  test  should  always  be  resorted  to  when  the 
copper  tests  give  a  positive  reaction  in  doubtful  cases. 

5.  Fermentation  Test. — This  is  simple  and  reliable,  but 
owing  to  the  time  required  it  is  not  much  used  in  routine 
work,  except  as  an  aid  in  distinguishing  dextrose  from 
other  forms  of  sugar.  It  is  carried  out  in  the  same  manner 
as  the  quantitative  test  (see  p.  169).  A  home-made 
device  which  answers  well  for  the  purpose  is  shown  in 
Fig.  44- 

Quantitative  Estimation. — In  quantitative  work  Feh- 
ling's  solution,  for  so  many  years  the  standard,  has  been 


1 66 


THE    URINE 


largely  displaced  by  Benedict's  quantitative  solution, 
which  appears  to  be  more  exact  and  more  satisfactory 
than  any  other  titration  method  available  for  sugar 
work.  The  older  method  is  still  preferred  by  some  and 
both  are  therefore  given. 

Should  the  urine  contain  much  glucose,  it  must  be 
diluted  before  making  any  quanti- 
tative test,  allowance  being  made 
for  the  dilution  in  the  subsequent 
calculation.  Albumin,  if  present, 
must  be  removed  by  acidifying  a 
considerable  quantity  of  urine  with 
acetic  acid,  boiling,  and  filtering. 
Any  water  lost  during  the  boiling 
should  be  replaced  before  filtering. 
A  rough  but  sometimes  useful 
approximation  of  the  amount  of 
sugar  in  the  urine  of  a  diabetic 
patient  can  be  made  by  estimating 
the  total  solids  (see  p.  no),  sub- 
tracting what  may  be  regarded  as 
Fig.     44— Simple     normal  for  the  individual  and  re- 

device  for  fermentation 

test  for  dextrose.  gardiug  the  remainder  as  sugar. 


I.  Fehlihg's  Method. — Take  lo  c.c.  of  Fehling's  solution 
(made  by  mixing  5  c.c.  each  of  the  copper  and  alkaline 
solutions  described  on  page  163)  in  a  flask  or  beaker,  add 
3  or  4  volumes  of  water,  boil,  and  add  the  urine  very  slowly 
from  a  buret  until  the  solution  is  completely  decolorized, 
heating  but  not  boiling  after  each  addition. 

Fehling's  Solution  is  of  such  strength  that  the  copper  in 
10  c.c.  will  be  reduced  by  exactly  0.05  Gm.  of  glucose. 
Therefore,  the  amount  of  urine  required  to  decolorize  the 


CHEMICAL   EXAMINATION  167 

test  solution  contains  just  0.05  Gm.  glucose,  and  the  per- 
centage is  easily  calculated. 

The  chief  objection  to  Fehling's  method  is  the  difficulty 
of  determining  the  end-point.  The  use  of  an  "outside  indi- 
cator," however,  obviates  this.  When  reduction  is  thought 
to  be  complete,  a  few  drops  of  the  solution  are  filtered 
through  a  fine-grained  filter-paper  on  to  a  porcelain  plate, 
quickly  acidified  with  acetic  acid,  and  mixed  with  a  drop  of 
10  per  cent,  potassium  ferrocyanid.  Immediate  appearance 
of  a  red-brown  color  shows  the  presence  of  unreduced 
copper. 

A  somewhat  simpler  application  of  this  method,  which  is 
accurate  enough  for  most  clinical  purposes,  is  as  follows: 
Take  i  c.c.  of  Fehling's  solution  in  a  large  test-tube,  dilute 
with  about  5  c.c.  of  water,  heat  to  boiling,  and,  while 
keeping  the  solution  hot  but  not  boiling,  add  the  urine 
drop  by  drop  from  a  medicine-dropper  until  the  blue 
color  is  entirely  gone.  Toward  the  end  add  the  drops  very 
slowly,  not  more  than  4  or  5  a  minute.  Divide  10  by  the 
number  of  drops  required  to  discharge  the  blue  color;  the 
quotient  wiil  be  the  percentage  of  glucose.  If  20  drops 
were  required,  the  percentage  would  be  10^20  =  0.5  per 
cent.  It  is  imperative  that  the  drops  be  of  such  size  that 
20  of  them  will  make  i  c.c.  Test  the  dropper  with  urine, 
not  water,  and  hold  it  always  at  the  angle  which  will  give 
the  right  sized  drop.  If  the  drops  are  too  large,  draw  out 
the  tip  of  the  dropper;  if  too  small,  cut  off  the  tip. 

2.  Benedict's  Method. — The  following  modification  of 
his  copper  solution  has  been  offered  by  Benedict  for  quanti- 
tative estimations. 

The  reagent  consists  of: 

Copper  sulphate  (pure  crystallized) 18.0  Gm.; 

Sodium  carbonate  (crystallized) 200.0  Gm.; 

(or  100  Gm.  of  the  anhydrous  salt). 

Sodium  or  potassium  citrate 200.0  Gm.; 


i68 


THE   URINE 


Potassium  sulphocyanate 125. oGm.; 

Potassium  f errocyanid  solution  (5  per  cent.) 5 .  o  c.c. ; 

Distilled  water,  to  make 1000. c 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate,  and 
sulphocyanate  in  about  700  c.c.  of  the  water  and  filter. 
Dissolve  the  copper  in  100  c.c.  of  water  and  pour  slowly 
into  the  other  fluid,  stirring  constantly.  Add  the  ferro- 
cyanid  solution,  cool,  and  dilute  to  1000  c.c.  Only  the 
copper  need  be  accurately  weighed.  This  solution  is  of  such 
strength  that  25  c.c.  are  reduced  by  0.05  Gm.  glucose.  It 
keeps  well. 


Fig.   45. — Einhorn's  sacchari meter. 

To  make  a  sugar  estimation,  take  25  c.c.  of  the  reagent  in 
a  small  flask,  add  10  to  20  Gm.  of  sodium  carbonate  crystals 
(or  one-half  this  weight  of  the  anhydrous  salt)  and  a  small 
quantity  of  powdered  pumice-stone  or  talcum.  Heat  to 
boiling,  and  add  the  urine  rather  rapidly  from  a  buret  until 
a  chalk-white  precipitate  forms  and  the  blue  color  of  the 
reagent  begins  to  fade.  After  this  point  is  reached,  add  the 
urine  a  few  drops  at  a  time  until  the  last  trace  of  blue  just 
disappears.     This  end-point  is  easily  recognized.     During 


CHEMICAL   EXAMINATION  1 69 

the  whole  of  the  titration  the  mixture  must  be  kept  vigor- 
ously boiling.  Loss  by  evaporation  must  be  made  up  by 
adding  water.  The  quantity  of  urine  required  to  discharge 
-the  blue  color  contains  exactly  0.05  Gm.  glucose,  and 
the  percentage  contained^  in  the  original  sample  is  easily  -< 
calculated.  ,  M>  ^     '   -V  X,o  h  .c^;,  (,c{  j^CA  ^.''"^-'-^Ij  ■ 

3.  Fermentation  Method. — This  k  convenient  and  sans- 
factory,  its  chief  disadvantage  being  the  time  required.  It 
depends  upon  the  fact  that  glucose  is  fermented  by  yeast 
with  evolution  of  CO2.  The  amount  of  gas  evolved  is  an 
index  of  the  amount  of  glucose.  No  preservative  must 
have  been  added.  Einhorn's  saccharimeter  (Fig.  45) 
is  the  simplest  apparatus. 

The  urine  must  be  so  diluted  as  to  contain  not  more  than 
I  per  cent,  of  glucose.  A  fragment  of  fresh  yeast-cake  about 
the  size  of  a  split-pea  is  mixed  with  a  definite  quantity  of  the 
urine  measured  in  the  tube  which  accompanies  the  ap- 
paratus. The  exact  amounts  of  yeast  and  urine  are  un- 
important. It  should  form  an  emulsion  free  from  lumps 
or  air-bubbles.  The  long  arm  of  the  apparatus  and  about 
half  the  bulb  are  then  filled  with  the  mixture,  all  bubbles 
being  carefully  discharged  by  tipping  the  instrument  with 
the  thumb  over  the  opening,  and  the  instrument  is  stood 
in  a  warm  place.  At  the  end  of  fifteen  to  twenty-four  hours 
fermentation  will  be  complete,  and  the  percentage  of  glu- 
cose can  be  read  off  upon  the  side  of  the  tube.  The  re- 
sult must  then  be  multiplied  by  the  degree  of  dilution. 
Since  yeast  itself  sometimes  gives  off  gas,  a  control  test 
must  be  carried  out  with  normal  urine  and  the  amount  of 
gas  evolved  must  be  subtracted  from  that  of  the  test.  A 
control  should  also  be  made  with  a  known  glucose  solution 
to  make  sure  that  the  yeast  is  active.  It  has  recently 
been  shown  that  yeast  can  split  off  carbon  dioxid  from 
amino-acids,  so  that  the  results  with  the  fermentation 
method  are  likely  to  be  a  little  high. 


170  THE    URINE 

The  method  is  not  applicable  to  urine  which  is  undergoing 
ammoniacal  fermentation. 

4.  Roberts'  Differential  Density  Method. — While  this 
method  gives  only  approximate  results,  it  is  convenient,  and 
requires  no  special  apparatus  but  an  accurate  urinometer. 
Mix  a  quarter  of  an  yeast-cake  with  about  100  c.c.  of  urine. 
Take  the  specific  gravity  and  record  it.  Set  the  urine  in  a 
warm  place  for  twenty-four  hours  or  until  fermentation  is 
complete.  Then  cool  to  the  temperature  at  which  the 
specific  gravity  was  originally  taken,  and  take  it  again. 
The  difference  between  the  two  readings  gives  the  number 
of  grains  of  sugar  per  ounce,  and  this,  multiplied  by  0.234, 
gives  the  percentage  of. sugar.  If  the  original  reading  is 
1.035,  ^^^  that  after  fermentation  is  1.020,  the  urine  con- 
tains 1.035  ~  I -020  =  15  gr.  of  sugar  per  fiuidounce;  and  the 
percentage  equals  15  X  0.234  =  3.5. 

(2)  Levulose,  or  fruit  sugar,  is  seldom  present  in 
urine  except  in  association  with  dextrose,*  and  has  about 
the  same  significance.  According  to  von  Noorden,  its 
appearance  in  diabetes  indicates  an  advanced  case.  Its 
name  is  derived  from  the  fact  that  it  rotates  polarized 
light  to  the  left. 

The  normal  assimilation  limit  for  levulose  is  about  100 
Gm.  This  fact  is  used  in  the  Strauss  test  of  the  func- 
tional capacity  of  the  liver.  One  hundred  grams  of 
levulose  are  given  upon  an  empty  stomach,  and  the 
subsequent  appearance  of  levulose  in  the  urine  is  taken 
as  evidence  of  deficiency  of  the  glycogenic  function. 
The  degree  of  the  hepatic  derangement  is  measured  by 
a  quantitative  estimation. 

Detection  of  Levulose. — Levulose  responds  to  all  the 
tests  above  given  for  dextrose.  It  may  be  distinguished 
from  dextrose  by  the  following: 


CHEMICAL    EXAMINATION  171 

Borchardt's  Test. — ^Mix  about  5  c.c.  each  of  the  urine  and 
25  per  cent,  hydrochloric  acid  (concentrated  HCl,  2  parts; 
water,  i  part)  in  a  test-tube  and  add  a  few  crystals  of  re- 
sorcinol.  Heat  to  boiling  and  boil  for  not  more  than  one- 
half  minute.  In  the  presence  of  levulose  a  red  color  appears. 
Cool  in  running  water,  pour  into  a  beaker,  and  render 
sHghtly  alkaline  with  solid  sodium  or  potassium  hydroxid. 
Return  to  the  test-tube,  add  2  or  3  c.c.  of  acetic  ether,  and 
shake.  If  levulose  be  present,  the  ether  will  be  colored 
yellow.  A  similar  yellow  color  will  follow  administration  of 
rhubarb  and  senna. 

If  indican  be  present  the  test  must  be  modified  as  follows: 
Perform  Obermayer's  test  and  extract  the  indican  with 
chloroform.  Reduce  the  acidity  of  the  indican-free  urine 
by  adding  one-third  its  volume  of  water,  add  a  few  crystals 
of  resorcinol,  and  proceed  with  Borchardt's  test. 

Quantitative  Estimation  of  Levulose. — The  methods 
are  the  same  as  for  dextrose  (see  p.  165).  Twenty- 
five  cubic  centimeters  of  Benedict's  quantitative  solu- 
tion are  reduced  by  0.053  Gm.  levulose. 

(3)  Lactose,  or  milk-sugar,  is  sometimes  present  in 
the  urine  of  nursing  women  and  in  that  of  women  who 
have  recently  miscarried.  It  is  of  interest  chiefly  be- 
cause it  may  be  mistaken  for  glucose.  //  reduces  copper, 
hut  does  not  ferment  with  yeast.  In  strong  solution  it  can 
form  crystals  with  phenylhydrazin,  but  is  extremely 
unlikely  to  do  so  when  the  test  is  applied  directly  to  the 
urine. 

(4)  Maltose  and  cane-sugar  are  of  little  or  no  clinical 
mportance.  Maltose  has  been  found  along  with  dex- 
trose in  diabetes.  It  reduces  copper,  0.074  Gm.  being 
equivalent  to  25  c.c.  of  Benedict's  solution.     Cane-sugar 


172  THE    URINE 

(sucrose)  is  sometimes  added  to  the  urine  by  malingering 
patients.     It  does  not  reduce  copper. 

(5)  Pentoses.- — These  sugars  are  so  named  because 
the  molecule  contains  5  atoms  of  carbon.  Vegetable 
gums  form  their  chief  source.  They  reduce  copper 
strongly  but  slowly,  and  give  crystals  with  phenyl- 
hydrazin,  but  do  not  ferment  with  yeast. 

Pentosuria  is  uncommon.  It  has  been  noted  after  in- 
gestion of  large  quantities  of  pentose-rich  substances, 
such  as  cherries,  plums,  and  fruit-juices,  and  is  said  to 
be  fairly  constant  in  habitual  use  of  morphin.  It  some- 
times accompanies  glycosuria  in  diabetes.  An  obscure 
chronic  form  of  pentosuria  without  clinical  symptoms 
has  been  observed.  The  pentose  excreted  in  these  cases 
is  believed  to  be  optically  inactive  arabinose,  although 
recent  work  indicates  that  ribose  is  present  in  some  cases 
at  least. 

Bial's  Orcinol  Test. — Dextrose  is  first  removed  by 
fermentation.  About  5  c.c.  of  Bial's  reagent  are  heated  in 
a  test-tube,  and  after  removing  from  the  flame  the  urine  is 
added  drop  by  drop,  not  exceeding  20  drops  in  all.  The 
appearance  of  a  green  color  denotes  pentose. 

The  reagent  consists  of: 

Hydrochloric  acid  (30  per  cent.) 500  c.c; 

Ferric  chlorid  solution  (10  per  cent.) 25  drops; 

Orcinol i  Gm. 

3.  Acetone  Bodies. — This  is  a  group  of  closely  re- 
lated substances — acetone,  diacetic  acid,  and  beta- 
oxybutyric  acid — whose  chief  source  is  in  abnormal 
katabolism  of  fats.  Formerly  beta-oxybutyric  acid 
was  held  to  be  the  mother  substance,  but  it  is  now  be- 


CHEMICAL   EXAMINATION  I73 

lieved  that  diacetic  acid  is  first  formed  and  that  the 
others  are  derived  from  it.  In  general,  their  presence 
in  the  urine  is  a  sign  of  acidosis  or  "acid-intoxication." 
When  the  disturbance  is  mild,  acetone  occurs  alone;  as 
it  becomes  more  marked,  diacetic  acid  is  also  found, 
and  finally  beta-oxybutyric  acid  appears. 

(i)  Acetone.— Minute  traces,  too  small  for  the  ordi- 
nary tests,  may  be  present  in  the  urine  under  normal 
conditions.  Larger  amounts  are  not  uncommon  when 
the  intake  of  carbohydrates  is  limited  and  in  fevers, 
gastro-intestinal  disturbances,  and  certain  nervous 
disorders.  A  notable  degree  of  acetonuria  has  likewise 
been  observed  in  pernicious  vomiting  of  pregnancy  and 
in  eclampsia. 

Acetonuria  is  practically  always  observed  in  acid 
intoxication,  and,  together  with  diaceturia,  constitutes 
its  most  significant  diagnostic  sign.  A  similar  or  identi- 
cal toxic  condition,  always  accompanied  by  acetonuria 
and  often  fatal,  is  now  recognized  as  a  not  infrequent 
late  effect  of  anesthesia,  particularly  of  chloroform  anes- 
thesia. This  postanesthetic  toxemia  is  more  likely  to 
appear,  and  is  more  severe,  when  the  urine  contains  any 
notable  amount  of  acetone  before  operation,  which  sug- 
gests the  importance  of  routine  examination  for  acetone 
in  surgical  cases. 

Acetone  is  present  in  considerable  amounts  in  many 
cases  of  diabetes  mellitus,  and  is  always  present  in  severe 
cases.  Its  amount  is  a  better  indication  of  the  severity 
of  the  disease  than  is  the  amount  of  sugar.  A  progres- 
sive increase  is  a  grave  prognostic  sign.  It  can  be  di- 
minished temporarily  by  more  liberal  allowance  of 
carbohydrates  in  the  diet,  by  addition  of  certain  vege- 


174 


THE   URINE 


tables  to  the  diet  (because  of  their  content  of  alkaline 
salts),  and  by  administration  of  bicarbonates. 

Acetonuria  from  any  cause  is  apt  to  be  especially 
marked  in  children,  and  this  doubtless  plays  an  impor- 
tant part  in  acute  and  chronic  diseases  of  childhood, 
especially  in  those  requiring  a  restricted  diet.  In  fact, 
the  urine  of  a  considerable  percentage  of  young  children 
shows  acetone  under  normal  conditions. 


Fig.  46. — A  simple  distilling  apparatus. 

According  to  Folin,  acetone  is  usually  present  in  only 
small  amounts  in  the  above  mentioned  conditions,  the 
substance  shown  by  the  usual  tests,  particularly  after 
distillation  of  the  urine,  being  really  diacetic  acid.  In 
this  connection,  Frommer's  test  is  to  be  recommended, 
since  it  does  not  require  distillation,  and  does  not  react 
to  diacetic  acid  unless  too  great  heat  is  applied. 

Detection  of  Acetone. — The  urine  may  be  tested  di- 


CHEMICAL   EXAMINATION  1 75 

rectly,  but  it  is  much  better  to  distil  it  after  adding  a 
little  phosphoric  or  hydrochloric  acid  to  prevent  foaming, 
and  to  test  the  first  few  cubic  centimeters  of  distillate. 
A  simple  distilling  apparatus  is  shown  in  Fig.  46.  The 
test-tube  may  be  attached  to  the  delivery  tube  by  means 
of  a  two-hole  rubber  cork  as  shown,  the  second  hole 
ser\nng  as  air  vent,  or,  what  is  much  less  satisfactory, 
it  may  be  tied  in  place  with  a  string.  Should  the  vapor 
not  condense  well,  the  test-tube  may  be  immersed  in  a 
glass  of  cold  water.  If  a  sufficiently  long  delivery  tube 
be  used  (2  feet)  there  will  be  little  difficulty  about 
condensation. 

When  diacetic  acid  is  present,  a  considerable  pro- 
portion will  be  converted  into  acetone  during  distilla- 
tion. 

Owing  to  the  marked  and  variable  loss  of  acetone 
through  the  lungs  a  quantitative  estimation  is  not  of 
much  value.  After  the  existence  of  an  acidosis  has 
been  established  by  the  detection  of  acetone  bodies,  it 
is  better  to  rely  upon  an  estimation  of  ammonia  as  a 
measure  of  its  severity. 

I.  Gunning's  Test. — To  about  five  cubic  centimeters  of 
urine  or  distillate  in  a  test-tube  add  5  drops  of  strong 
ammonia  and  then  Lugol's  solution  in  sufficient  quantity 
to  produce  a  black  cloud  which  does  not  immediately 
disappear.  This  cloud  will  gradually  clear  up  and,  if 
acetone  be  present,  iodoform,  usually  crystalline,  will 
separate  out.  The  iodoform  can  be  recognized  by  its  odor, 
especially  upon  heating  (there  is  danger  of  explosion  if  the 
mixture  be  heated  before  the  black  cloud  disappears), 
or  by  detection  of  the  crystals  microscopically.  The 
latter,    alone,    is    dependable,    unless    one    has   an   acute 


176  THE   URINE 

sense  of  smell.  The  odor  of  iodin,  which  is  also  present, 
is  often  confusing.  Iodoform  crystals  are  yellowish  six- 
pointed  stars  or  six-sided  plates  (Fig.  47). 

This  modification  of  Lieben's  test  is  less  sensitive  than  the 
original,  but  is  sufficient  for  all  clinical  work;  it  has  the  ad- 
vantage that  alcohol  does  not  cause  confusion,  and  especially 
that  the  sediment  of  iodoform  is  practically  always  crys- 
talline. When  it  is  applied  directly  to  the  urine,  phos- 
phates are  precipitated  and  may  form  large  feathery,  star- 


rv  ii/fiJKtg.'*  I  IB*  c-?*«w^r"*wv«n.  .•.rij"««cr~v'~T-TrT 


^^t>(si^- 


Fig.  47. — Iodoform  crystals  obtained  in  several  tests  for  acetone  by 
Gunning's  method  (  X  about  600). 


shaped  crystals  which  are  confusing  to  the  inexperienced 
(see  Fig.  56).  Albumin  prevents  formation  of  the  crystals, 
and  when  it  is  present,  the  urine  must  be  distilled  for  the 
test. 

2.  Lange's  Test. — This  is  a  modification  of  the  well- 
known  Legal  test.  It  is  more  sensitive  and  gives  a  sharper 
end-reaction.  To  a  small  quantity  of  urine  add  about  one- 
twentieth  its  volume  (i  drop  for  each  i  c.c.)  of  glacial  acetic 
acid  and  a  few  drops  of  fresh  concentrated  aqueous  solution 
of  sodium  nitroprussid,  and  gently  run  a  little  ammonia 
upon  its  surface.     If  acetone  be  present,  a  reddish-purple 


CHEMICAL   EXAMINATION  1 77 

ring  will  form  within  a  few  minutes  at  the  junction  of  the 
two  fluids. 

Lange's  test  is  even  more  sensitive  to  diacetic  acid  than 
to  acetone.  For  this  reason,  Rothera's  test,  which  is  more 
sensitive  to  acetone,  is  to  be  preferred:  To  5  or  10  c.c. 
of  urine  add  about  a  gram  of  ammonium  sulphate,  and  2 
or3  drops  of  fresh  concentrated  sodium  nitroprussid  so- 
lution and  overlay  with  strong  ammonia.  A  permanganate 
colored  ring  shows  the  presence  of  acetone. 

3.  Frommer's  Test. — This  test  has  proved  very  satis- 
factory in  the  hands  of  the  writer.  The  urine  need  not  be 
distilled.  Alkalinize  about  10  c.c.  of  the  urine  with  2  or 
3  c.c.  of  40  per  cent,  caustic  soda  solution,  add  10  or  12 
drops  of  10  per  cent,  alcoholic  solution  of  salicylous  acid 
(salicyl  aldehyd),  heat  the  upper  portion  to  about  7o°C. 
(it  should  not  reach  the  boiling-point),  and  keep  at  this 
temperature  five  minutes  or  longer.  In  the  presence  of 
acetone  an  orange  color,  changing  to  deep  red,  appears  in 
the  heated  portion  A  yellow  to  brown  color  may  appear 
in  the  absence  of  acetone. 

The  test  can  be  made  more  definite  by  adding  the  caustic 
soda  in  substance  (about  i  Gm.),  and  before  it  goes  into 
solution  adding  the  salicyl  aldehyd  and  warming  the  lower 
portion. 

(2)  Diacetic  (aceto-acetic)  acid  occurs  in  the  same 
conditions  as  acetone,  but  has  more  serious  significance. 
In  diabetes  its  presence  is  a  grave  symptom  and  often 
forewarns  of  approaching  coma.  It  rarely  or  never 
occurs  without  acetone. 

Detection. — The  urine  must  be  fresh.  If  a  preserva- 
tive must  be  used,  toluene  is  best. 

Gerhardt's  Test. — ^To  a  few  cubic  centimeters  of  the 
urine  add  solution  of  ferric  chlorid  (about  10  per  cent.) 

12 


178  THE    URINE 

drop  by  drop  until  the  phosphates  are  precipitated;  filter 
and  add  more  of  the  ferric  chlorid.  If  diacetic  acid  be 
present,  the  urine  will  assume  a  Bordeaux-red  color  which 
disappears  upon  boiling.  Several  minutes  boiling  are 
required;  simply  bringing  the  fluid  to  the  boiling-point  will 
not  suffice.  A  red  or  violet  color  which  does  not  disappear 
upon  boiling  may  be  produced  by  other  substances,  as 
phenol,  salicylates,  and  antipyrin.  Whenever  the  reaction 
is  doubtful  the  urine  should  be  distilled  and  the  distillate 
tested  for  acetone. 

The  test  is  somewhat  more  definite  if  applied  by  the 
contact  or  "ring"  method  (see  p.  152). 

(3)  Oxybutyric  acid  has  much  the  same  significance 
as  diacetic  acid,  but  is  of  more  serious  import. 

Hart's  Test. — Remove  acetone  and  diacetic  acid  by  dilut- 
ing 20  c.c.  urine  with  20  c.c.  water,  adding  a  few  drops  of 
acetic  acid,  and  boiling  down  to  10  c.c.  To  this  add  10  c.c. 
water,  mix,  and  divide  between  two  test-tubes.  To  one 
tube  add  i  c.c.  of  hydrogen  peroxid,  warm  gently,  and  cool. 
This  transforms  ^-oxybutyric  acid  to  acetone.  Now  apply 
Lange's  test  for  acetone  (see  p.  176)  to  each  tube.  A 
positive  reaction  in  the  tube  to  which  hydrogen  peroxid 
has  been  added  shows  the  presence  of  ^-oxybutyric  acid 
in  the  original  sample  of  urine. 

4.  Bile. — The  pigment  of  bile  has  its  origin  in  the 
never-ceasing  destruction  of  red  blood-corpuscles  within 
the  body. 

The  significance  of  bile  in  the  urine  is  practically 
the  same  as  that  of  bile-staining  of  the  tissues,  known  as 
icterus  or  jaundice.  Small  amounts  of  bile  may,  how- 
ever, be  found  in  the  urine  when  the  disturbance  is 
not  severe  enough  to  produce  recognizable  jaundice 


CHEMICAL   EXAMINATION  1 79 

or,  in  other  cases,  before  the  jaundice  supervenes.  The 
usual  cause  of  icterus  is  some  obstruction  to  the  out- 
flow of  bile  from  the  liver,  which  may  be  in  the  nature  of 
foreign  bodies  or  new  growths  inside  or  outside  of  the 
bile-passages,  or  inflammatory  swelling  of  the  walls 
with  narrowing  of  the  lumen.  Jaundice  may  also  occur 
when  there  is  excessive  destruction  of  red  blood-cor- 
puscles from  any  cause.  This  leads  to  excessive  for- 
mation of  a  bile  which  is  more  inspissated  than  normal 
and  thus  tends  to  block  the  bile-capillaries.  Another, 
less  frequent,  cause  is  rapid  destruction  of  liver  cells  as 
in  acute  yellow  atrophy  and  phosphorus  poisoning. 
Strong  emotion  has  also  been  known  to  cause  jaundice  in 
some  obscure  way.  Both  bile-pigment  and  bile  acids 
may  be  found.  They  generally  occur  together,  but 
the  pigment  is  not  infrequently  present  alone. 

Of  the  several  pigments  bilirubin,  alone,  occurs  in 
freshly  voided  urine,  the  others  (biliverdin,  bilifuscin, 
etc.)  being  produced  from  this  by  oxidation  as  the  urine 
stands.  The  acids,  which  occur  chiefly  as  sodium  salts, 
are  almost  never  present  without  the  pigments,  and  are, 
therefore,  seldom  tested  for  clinically.  Crystals  of 
bilirubin  (hematoidin)  (Fig.  49,  4)  may  be  deposited  in 
heavily  bile-charged  urine. 

Detection  of  Bile -pigment. — Bile-pigment  gives  the 
urine  a  greenish-yellow,  yellow,  or  brown  color,  which 
upon  shaking  is  imparted  to  the  foam.  Cells,  casts,  and 
other  structures  in  the  sediment  may  be  stained  brown 
or  yellow.  This,  however,  should  not  be  accepted  as 
proving  the  presence  of  bile  without  further  tests. 

I.  Smith's  Test. — Overlay  the  urine  with  tincture  of 
iodin  diluted  with  nine  times  its  volume  of  alcohol.     An 


l8o  THE    UBINE 

emerald-green  ring  at  the  zone  of  contact  shows  the  presence 
of  bile-pigments.  It  is  convenient  to  use  a  conical  test- 
glass,  one  side  of  which  is  painted  white. 

2.  Gmelin's  Test. — This  consists  in  bringing  slightly 
yellow  nitric  acid  into  contact  with  the  urine.  A  play  of 
colors,  of  which  green  and  violet  are  most  distinctive, 
denotes  the  presence  of  bile-pigment.  Blue  and  red  may 
be  produced  by  indican  and  urobilin.  Colorless  nitric 
acid  will  become  yellow  upon  standing  in  the  sunlight. 
The  test  may  be  applied  in  various  ways:  by  overlaying 
the  acid  with  the  urine;  by  bringing  a  drop  of  each  together 
upon  a  porcelain  plate;  by  filtering  the  urine  through  thick 
filter-paper,  and  touching  the  paper  with  a  drop  of  the  acid ; 
and,  probably  best  of  all,  by  precipitating  with  lime-water, 
filtering,  and  touching  the  precipitate  with  a  drop  of  the 
acid.  In  the  last  method  bilirubin  is  carried  down  as  an 
insoluble  calcium  compound  which  concentrates  the  pig- 
ment and  avoids  interfering  substances. 

Detection  of  Bile  Acids. — Hay's  test  is  simple,  sensi- 
tive, and  fairly  reliable,  and  will,  therefore,  appeal  to 
the  practitioner.  It  depends  upon  the  fact  that  bile 
acids  lower  surface  tension.  Other  tests  require  isola- 
tion of  the  acids  for  any  degree  of  accuracy. 

Hay's  Test. — Upon  the  surface  of  the  urine,  which  must 
not  he  warm,  sprinkle  a  little  finely  powdered  sulphur 
("flowers  of  sulphur").  If  it  sink?  at  once,  bile  acids  are 
present  to  the  amount  of  o.oi  per  cent,  or  more;  if  only 
after  gentle  shaking,  0.0025  per  cent,  or  more.  If  it  re- 
mains floating,  even  after  gentle  shaking,  bile  acids  are 
absent.  It  is  said  that  urobilin  when  present  in  large 
amount  also  reduces  surface  tension. 

5.  Hemoglobin. — The  presence  in  the  urine  of 
hemoglobin  or  pigments  directly  derived  from  it,  ac- 


CHEMICAL  EXAMINATION  l8l 

companied  by  few,  if  any,  red  corpuscles,  constitutes 
hemoglobinuria.  It  is  a  comparatively  rare  condition, 
and  must  be  distinguished  from  hematuria,  or  blood  in 
the  urine,  which  is  common.  In  both  conditions  chemic 
tests  will  show  hemoglobin,  but  in  the  latter  the  micro- 
scope will  reveal  the  presence  of  red  corpuscles.  Urines 
which  contain  notable  amounts  of  hemoglobin  have  a 
reddish  or  brown  color,  and  may  deposit  a  sediment  of 
brown,  granular  pigment. 

Hemoglobinuria  occurs  when  there  is  such  extensive 
destruction  of  red  blood-cells  within  the  body  that  the 
liver  cannot  transform  all  the  hemoglobin  set  free  into 
bile-pigment.  The  most  important  examples  are  seen 
following  extensive  burns  in  poisoning,  as  by  mushrooms 
and  potassium  chlorate,  in  scurvy  and  purpura,  in 
malignant  malaria  (black water  fever) ,  and  in  the  obscure 
condition  known  as  ''paroxysmal  hemoglobinuria." 
This  last  is  characterized  by  the  appearance  of  large 
quantities  of  hemoglobin  at  intervals,  usually  following 
exposure  to  cold,  the  urine  remaining  free  from  hemo- 
globin between  the  attacks. 

Detection.— Teichmann's  test  may  be'  applied  to  the 
precipitate  after  boiling  and  filtering,  but  this  is  not 
very  satisfactory,  and  the  guaiac  or  benzidin  test  will  be 
found  more  convenient  in  routine  work.  For  further 
discussion  of  blood  tests,  including  spectroscopic  meth- 
ods, see  page  364. 

Guaiac  Test. — Mix  a  few  cubic  centimeters  each  of 
"ozonized"  turpentine  and  a  fresh  i  : 60  alcoholic  solution 
of  guaiac.  The  guaiac  solution  may  be  freshly  prepared  by 
dissolving  a  pocket-knife-pointful  of  powdered  guaiac  in 
4  or  5  CO.  of  alcohol.     Carry  out  the  test  by  the  "  ring  " 


1 82  THE    URINE 

or  "contact"  method  described  on  page  152,  stratifying 
the  guaiac-turpentine  mixture  upon  the  urine.  A  bright 
blue  ring  will  appear  at  the  zone  of  contact  \vithin  a  few 
minutes  if  hemoglobin  be  present.  The  guaiac  should  be 
kept  in  an  amber-colored  bottle.  Fresh  turpentine  can 
be  "ozonized"  by  allowing  it  to  stand  a  few  days  in  an 
open  vessel  in  the  sunlight.  Instead  of  turpentine,  hy- 
drogen peroxid  may  be  used. 

This  test  is  very  sensitive,  and  a  negative  result  proves  the 
absence  of  hemoglobin.  Positive  results  are  not  con- 
clusive, because  numerous  other  substances — few  of  them 
likely  to  be  found  in  the  urine — may  produce  the  blue 
color.  That  most  Ukely  to  cause  confusion  is  pus,  but 
the  blue  color  produced  by  it  disappears  upon  heating 
and  will  appear  equally  well  if  the  turpentine  be  omitted. 
The  thin  film  of  copper  often  left  in  a  test-tube  after  test- 
ing for  sugar  may  give  the  reaction,  as  may  also  the  fumes 
from  an  open  bottle  of  bromin.  Most  sources  of  error 
can  be  avoided  by  extracting  the  hemoglobin  with  ether  as 
described  on  page  364. 

Benzidin  Test. — The  reagents  employed  are  hydrogen 
peroxid  and  a  saturated  solution  of  benzidin  in  glacial  acetic 
acid.  The  benzidin  labeled  "For  blood  tests"  should  be 
employed.  The  reagents  are  mixed  in  equal  parts  and  a 
few  cubic  centimeters  are  added  to  a  like  amount  of  the 
urine.  A  blue  color  appears  in  the  presence  of  hemoglobin. 
The  test  has  the  same  fallacies  as  the  guaiac  test,  but  is 
more  sensitive  and  in  general  more  satisfactory. 

The  benzidin  test  may  be  simplified  by  use  of  the 
tablets  recently  put  upon  the  market  by  the  firm  of 
E.  R.  Squibb  &  Sons.  These  contain  benzidin  and 
sodium  perborate.  A  tablet  is  thoroughly  moistened  with 
the  fluid  to  be  tested  and  is  then  touched  with  a  drop  of 
glacial  acetic  acid,  the  appearance  of  a  blue  color  indicat- 
ing blood.     The  test  is  less  delicate  than  those  given  above. 


CHEMICAL    EXAMINATION  1 83 

Spectroscopic  Method. — This  is  discussed  on  page  366. 
It  is  more  reliable  than  the  preceding  tests  but  less  sensitive. 
Render  the  urine  slightly  acid,  dilute  if  very  highly  colored 
and  examine  with  a  small  direct-vision  spectroscope.  The 
usual  bands  seen  are  those  of  oxyhemoglobin  and  met- 
hemoglobin. 

To  detect  traces  of  blood  proceed  as  described  in  section 
2  on  page  368.  This  method  will  easily  detect  2  drops 
of  blood  in  8  ounces  of  urine. 

6.  Alkapton  Bodies. — ^The  name  "alkaptonuria" 
has  been  given  to  a  condition  in  which  the  urine  turns 
reddish  brown  to  brownish  black  upon  standing  and 
strongly  reduces  copper  (but  not  bismuth),  owing  to  the 
presence  of  certain  substances  which  result  from  imper- 
fect protein  metabolism.  The  chief  of  these  is  homo- 
gentisic  acid.  The  change  of  color  takes  place  quickly 
when  fresh  urine  is  alkalinized,  hence  the  name,  alkapton 
bodies. 

Alkaptonuria  is  unaccompanied  by  other  symptoms, 
and  has  little  clinical  importance.  Only  a  few  cases, 
mostly  congenital,  have  been  reported.  The  change  in 
color  of  the  urine  and  the  reduction  of  copper  with  no 
reduction  of  bismuth  nor  fermentation  with  yeast  would 
suggest  the  condition. 

7.  Melanin. — Urine  which  contains  melanin  likewise 
darkens  upon  exposure  to  the  air,  assuming  a  dark 
brown  or  black  color.  This  is  due  to  the  fact  that  the 
substance  is  eliminated  as  a  chromogen- — melanogen — 
which  is  later  converted  into  the  pigment.  It  does  not 
reduce  copper. 

Melanuria  occurs  in  most,  but  not  all,  cases  of  mela- 
notic tumor.     Its  diagnostic  value  is  lessened  by  the 


184  THE    URINE 

fact    that    it    has    been    observed    in   other   wasting 
diseases. 

Tests  for  Melanin. — i.  Addition  of  ferric  chlorid  gives  a 
gray  precipitate  which  blackens  on  standing. 

2.  Bromin  water  causes  a  yellow  precipitate  which 
gradually  turns  black. 

8.  Hematoporphyrin  is  an  iron-free  pigment  de- 
rived from  hemoglobin.  Its  formation  within  the  body 
is  not  well  understood.  Normally  it  appears  in  the 
urine  only  in  slight  traces.  An  increase  has  been  ob- 
served in  a  variety  of  conditions,  notably  in  organic 
liver  diseases,  in  lead-poisoning,  and,  especially,  during 
continued  use  of  sulphonal,  trional  and  tetronal.  In 
the  presence  of  abnormal  amounts  the  urine  may  have 
a  dark  red  or  "port  wine"  color,  which,  however,  ap- 
pears to  be  due  in  part  to  the  simultaneous  presence  of 
related  but  little-known  pigments. 

Hematoporphyrin  does  not  respond  to  the  guaiac  or  the 
hemin  test  and  is  best  detected  with  the  spectroscope. 
Treat  100  c.c.  of  the  urine  with  20  c.c.  of  10  per  cent, 
sodium  hydroxid  solution.  The  pigment  will  be  carried 
down  with  the  phosphates.  Filter  (or  centrifugalize), 
wash  the  precipitate  with  water  then  with  alcohol,  and 
finally  dissolve  in  about  5  c.c.  of  alcohol  to  which  5  to  10 
drops  of  concentrated  hydrochloric  acid  have  been  added. 
Examine  the  acid  solution  for  the  absorption  bands  of 
acid  hematoporphyrin  (Fig.  142). 

9,  Urobilin. — Traces  of  this  pigment,  too  small  for 
detection  by  the  ordinary  tests,  are  present  under  nor- 
mal conditions.  It  is  now  regarded  as  identical  with 
hydrobilirubin,   the   principal   coloring  matter  of   the 


CHEMICAL   EXAMINATION  185 

feces.  It  is  excreted  as  a  chromogen,  urobilinogen, 
which  is  changed  into  urobiHn  through  the  action  of 
light  within  a  few  hours  after  the  urine  is  voided.  A 
great  excess  gives  the  urine  a  dark  brown  color  suggest- 
ing the  presence  of  bile,  but  does  not  color  the  foam 
as  does  bile.  Small  amounts  cause  no  perceptible 
change  in  color. 

The  mode  of  formation  of  urobilin  is  not  yet  clearly 
understood.  According  to  the  generally  accepted  view 
it  is  a  decomposition  product  of  bilirubin,  formed 
chiefly  in  the  intestine  through  the  action  of  bacteria. 
Upon  the  other  hand,  the  formation  of  small  amounts  in 
the  liver  itself  under  certain  conditions  cannot  be 
denied.  Urobilinogen  is  first  formed.  Under  normal 
conditions  a  portion  of  this  is  absorbed  from  the  intes- 
tine, carried  to  the  liver  in  the  portal  blood,  and  there 
reconverted  into  bilirubin.  When  the  liver  cells  are 
deranged,  this  transformation  into  bilirubin  does 
not  take  place  and  urobilinogen  reaches  the  general 
circulation  and  is  excreted  by  the  kidneys.  The  re- 
mainder in  the  intestine,  changed  largely  into  urobilin, 
passes  out  with  the  feces.  The  pigment  and  the 
chromogen  have  exactly  the  same  significance  in  the 
urine,  and  the  name  "urobilin"  is  commonly  used  to 
cover  both. 

Owing  to  the  many  unknown  factors  it  is  impossible 
to  ascribe  definite  clinical  significance  to  urobilinuria. 
Certain  statements,  however,  seem  justified.  When- 
ever, owing  to  excessive  destruction  of  blood-corpuscles, 
there  is  excessive  formation  of  bilirubin  and  hence  an 
increase  of  urobilin  in  the  feces,  there  is  also  a  marked 
increase  in  the  urine.     With  this  exception,  urobilinuria 


1 86  THE    URINE 

usually  points  toward  functional  incapacity  of  the  liver. 
Its  recognition  is  very  simple  and  has  considerable 
practical  usefulness,  as  for  example  in  the  diagnosis  of 
hepatic  cirrhosis;  in  judging  the  amount  of  damage  done 
to  the  liver  parenchyma  by  poisons  and  the  chronic 
congestion  of  poorly  compensated  heart  disease;  and  in 
differentiating  anemias  associated  with  excessive  blood 
destruction  {e.g.,  pernicious  anemia)  from  those  due  to 
other  causes  (carcinoma,  hemorrhage).  Urobilinuria 
is  frequent  in  acute  infectious  diseases,  especially  in 
scarlet  fever  and  pneumonia,  and  usually  means  either 
hemolysis  or  damage  to  the  liver.  In  severe  nephritis 
urobilin  may  fail  to  be  excreted  even  when  other  condi- 
tions favor  its  appearance  in  the  urine.  It  is  nearly  or 
entirely  absent  in  obstructive  jaundice. 

To  be  of  value,  tests  for  urobilin  should  be  made  upon 
several  successive  days,  because  for  some  unknown 
reason  it  may  be  absent  for  a  day  or  two,  and  it  is 
advisable  to  make  a  simultaneous  study  of  the  urobilin 
of  the  feces. 

1.  Ehrlich's  Test  for  Urobilinogen. — To  a  few  cubic 
centimeters  of  the  urine  in  a  test-tube  add  a  few  crystals  of 
para-dimethyl-amino-benzaldehyd  and  make  definitely  acid 
with  hydrochloric  acid.  In  the  presence  of  pathologic 
amounts  of  urobilinogen  a  cherry-red  color  appears.  This 
is  best  seen  by  viewing  the  tube  from  the  top  over  a  sheet  of 
white  paper.  Normal  amounts  will  cause  the  red  color 
only  when  the  urine  is  heated. 

2.  Schlesinger's  Test  for  Urobilin. — To  about  5  c.c.  of 
the  urine  in  a  test-tube  add  a  few  drops  of  Lugol's  solution 
to  transform  the  chromogen  into  the  pigment.  Now  add 
4  or  5  c.c.  of  a  saturated  alcoholic  solution  of  zinc  acetate 


CHEMICAL   EXAMINATION  187 

or  zinc  chlorid  and  filter.  A  greenish  fluorescence,  best 
seen  when  the  tube  is  viewed  in  bright  sunlight  against  a 
black  background  and  when  the  light  is  concentrated  upon  it 
with  a  lens,  shows  the  presence  of  urobilin.  The  fluores- 
cence becomes  more  marked  after  an  hour  or  two.  Bile- 
pigment,  if  present,  should  be  previously  removed  by 
adding  about  one-fifth  volume  of  10  per  cent,  calcium  chlorid 
solution  and  filtering. 

Quantitative  Estimation. — The  indirect  although 
clinically  satisfactory  method  of  Wilber  and  Addis 
which  is  given  in  detail  on  page  432  may  be  applied  to 
the  urine  as  follows: 

1.  To  a  lo-c.c.  portion  of  the  mixed  twenty-four-hour 
urine,  which  has  been  preserved  with  a  crystal  of  thymol  and 
kept  in  darkness,  add  10  c.c.  of  a  saturated  alcoholic 
solution  of  zinc  acetate  and  filter. 

2.  To  10  c.c.  of  the  filtrate  (representing  5  c.c.  of  urine) 
add  I  c.c.  of  Ehrlich's  reagent,  the  formula  for  which  is 
as  follows: 

Para-dimethyl-amino-benzaldehyd 10  gm. 

Concentrated  hydrochloric  acid 75  c.c. 

Water 75  c.c. 

3.  Let  stand  in  the  dark  for  one  to  three  hours,  not  longer. 

4.  Examine  with  a  small  direct-vision  spectroscope  and 
dilute  with  tap  water  until  absorption  bands  disappear. 
Calculate  the  total  dilution  value  for  the  twenty-four- 
hour  quantity  of  urine  as  described  for  urobilin  in  feces, 
basing  the  calculation  upon  the  5  c.c.  of  urine  used. 

Example. — If  the  twenty-four-hour  urine  amounts  to 
1200  c.c.  and  it  is  necessary  to  dilute  the  lo-c.c.  filtrate  to 
50  c.c.  to  get  rid  of  the  absorption  bands  (supposing  that 
they  disappear  together),  then  the  combined  dilution  value 


1 88  THE   URINE 

for  urobilin  and  urobilinogen  in  the  5  c.c.  of  urine  would  be 
10  +  10  =  20;  and  the  twenty-four-hour  value  would 
be  20  X  240  =  4800. 

10.  Diazo  Substances. — Certain  imperfectly  known 
substances  sometimes  present  in  the  urine  give  a 
characteristic  color  reaction — the  "diazo-reaction"  of 
Ehrlich — when  treated  with  diazo-benzol-sulphonic 
acid  and  ammonia.  This  reaction  has  much  clinical 
value,  provided  its  limitations  he  recognized.  It  is  at 
best  an  empirical  test  and  must  be  interpreted  in  the 
light  of  clinical  symptoms.  Although  it  has  been  met 
with  in  a  considerable  number  of  diseases,  its  usefulness 
is  practically  limited  to  typhoid  fever,  tuberculosis,  and 
measles. 

(i)  Typhoid  Fever. — Practically  all  cases  give  a 
positive  reaction,  which  varies  in  intensity  with  the 
severity  of  the  disease.  It  is  so  constantly  present  that 
it  is  sometimes  said  to  be  "negatively  pathognomonic:" 
if  negative  upon  several  successive  days  at  a  stage  of  the 
disease  when  it  should  he  positive,  typhoid  is  almost 
certainly  absent.  Upon  the  other  hand,  a  reaction 
when  the  urine  is  highly  diluted  (1:50  or  more)  has 
much  positive  diagnostic  value,  since  this  dilution  pre- 
vents the  reaction  in  most  conditions  which  might  be 
mistaken  for  typhoid;  but  it  should  be  noted  that  mild 
cases  of  typhoid  may  not  give  it  at  this  dilution.  Ordi- 
narily the  diazo-  appears  a  little  earlier  than  the  Widal 
reaction — about  the  fourth  or  fifth  day — but  it  may  be 
delayed.  In  contrast  to  the  Widal,  it  begins  to  fade 
about  the  end  of  the  second  week,  and  soon  thereafter 
entirely  disappears.  An  early  disappearance  is  a  favor- 
able sign.     It  reappears  during  a  relapse,  and  thus  helps 


CHEMICAL   EXAMINATION  '  1 89 

to  distinguish  between  a  relapse  and  a  complication, 
in  which  it  does  not  reappear. 

(2)  Tuberculosis.— The  diazo-reaction  has  been  ob- 
tained in  many  forms  of  the  disease.  It  has  little  or 
no  diagnostic  value.  Its  continued  presence  in  pul- 
monary tuberculosis  is,  however,  a  grave  prognostic 
sign,  even  when  the  physical  signs  are  slight.  After  it 
once  appears  it  generally  persists  more  or  less  intermit- 
tently until  death,  the  average  length  of  life  after  its 
appearance  being  about  six  months.  The  reaction  is 
often  temporarily  present  in  mild  cases  during  febrile 
complications,  and  has  then  no  significance. 

(3)  Measles. — A  positive  reaction  is  usually  obtained 
in  measles,  and  may  help  to  distinguish  this  disease 
from  German  measles,  in  which  it  does  not  occur.  It 
generally  appears  before  the  eruption  and  remains  about 
five  days. 

Technic. — Although  the  test  is  really  a  very  simple  one, 
careful  attention  to  technic  is  imperative.  Many  of  the 
early  workers  were  very  lax  in  this  regard.  Faulty  technic 
and  failure  to  record  the  stage  of  the  disease  in  which  the 
tests  were  made  have  probably  been  responsible  for  the 
bulk  of  the  conflicting  results  reported. 

Certain  drugs  often  given  in  tuberculosis  and  typhoid 
interfere  with  the  reaction  or  prevent  it.  The  chief  are 
creosote,  tannic  acid  and  its  compounds,  opium  and  its 
alkaloids,  salol,  phenol,  and  the  iodids.     The  reagents  are: 

(i)  Sulphanilic  acid i  Gm.; 

Concentrated  hydrochloric  acid 10  c.c; 

Water 200  c.c; 

(2)  Sodium  nitrite 0.5  Gm.; 

Water 100  c.c. 

(3)  Strong  ammonia. 


190  THE    URINE 

Mix  icx)  parts  of  (i)  and  i  part  of  (2).^  In  a  test-tube 
take  equal  parts  of  this  mixture  and  the  urine,  and  pour  i  or 
2  c.c.  of  the  ammonia  upon  its  surface.  If  the  reaction  be 
positive,  a  garnet  ring  will  form  at  the  junction  of  the  two 
fluids;  and,  upon  shaking,  a  distinct  pink  color  will  be  im- 
parted to  the  foam.  The  color  of  the  foam  is  the  essential 
feature.  If  desired,  the  mixture  may  be  well  shaken  before 
the  ammonia  is  added:  the  pink  color  will  then  instantly 
appear  in  that  portion  of  the  foam  which  the  ammonia  has 
reached,  and  can  be  readily  seen.  The  color  varies  from 
eosin-pink  to  deep  crimson,  depending  upon  the  intensity 
of  the  reaction.  It  is  a  pure  pink  or  red;  any  trace  of  yellow 
or  orange  denotes  a  negative  reaction.  A  doubtful  reaction 
should  be  considered  negative. 

Substitutes  for  the  Diazo-reaction. — The  two  follow- 
ing tests,  which  have  been  oflfered  as  simple  and  satis- 
factory substitutes  for  the  diazo,  have  found  rather 
wide  acceptance.  They  are  supposed  to  be  positive  in 
the  same  classes  of  cases  as  the  diazo  and  to  have  the 
same  clinical  significance,  but  are  claimed  to  be  more 
reliable. 

I.  Weis's  Urochromogen  Test. — In  a  test-tube  mix  2  c.c. 
of  urine  and  4  c.c.  distilled  water,  and  add  3  drops  of  i :  1000 
aqueous  solution  of  potassium  permanganate.  The  appear- 
ance of  a  yellow  color  denotes  a  positive  reaction.  The 
color  is  best  judged  by  comparison  with  a  tube  of  diluted 
urine  to  which  no  permanganate  has  been  added,  the  two 
tubes  being  viewed  from  the  top  over  a  sheet  of  white 
paper.  The  color  of  a  genuine  reaction  is  a  canary  yel- 
low. A  yellow  color,  usually  not  so  bright,  and  tending 
more   toward   brown,  may  be  produced  by  urobilin  and 

^  These  proportions  are  recommended  by  Greene,  and  are  now  gen- 
erally used.     Ehrlich  used  40  parts  of  (i)  and  i  part  of  (2). 


CHEMICAL   EXAMINATION  IQI 

Other  substances,  but  these  false  reactions  fade  quickly, 
usually  within  thirty  seconds,  while  the  color  of  a  true 
reaction  remains  a  longer  time. 

Weis  believes  the  diazo-reaction  to  be  due  principally  to 
urochromogen,  which,  because  of  the  effect  of  certain  toxins 
upon  metabolism,  fails  of  conversion  into  urochrome;  and 
he  has  offered  this  permanganate  reaction  as  a  more 
satisfactory  test,  both  for  urochromogen  and  for  an  ante- 
cedent substance  which  has  the  same  significance  as  uro- 
chromogen, but  which  the  diazo  fails  to  detect.  This  test 
has  been  studied  chiefly  in  its  relation  to  prognosis  in 
tuberculosis,  in  which  it  appears  to  have  about  the  same 
value  as  the  diazo,  with  the  differences  that  it  is  more 
frequently  noted  and  is  less  intermittent  in  a  given  case 
and  probably  has  less  serious  import. 

2.  Russo's  Methylene-blue  Test. — To  5  c.c.  of  the  urine 
in  a  test-tube  add  5  drops  of  i :  1000  aqueous  solution  of 
methylene-blue,  and  mix.  An  emerald-  or  mint-green  color, 
in  which  there  must  be  no  trace  of  blue,  denotes  a  positive 
reaction.  There  is  considerable  difficulty  in  judging  the 
color. 

Since  this  test  was  offered  in  1905,  itiias  been  condemned 
by  many  workers  and  extolled  by  others.  In  the  writer's 
opinion  it  is  worthless  for  the  purpose  for  which  it  was 
offered.  At  most,  it  is  a  very  rough  quantitative  test  for 
urochrome. 

11.  Drugs. — The  effect  of  various  drugs  upon  the 
color  of  the  urine  has  been  mentioned  (see  p.  103).  Most 
poisons  are  eliminated  in  the  urine,  but  their  detection 
is  more  properly  discussed  in  works  upon  toxicology.  A 
few  drugs  which  are  of  interest  to  the  practitioner,  and 
which  can  be  detected  by  comparatively  simple  methods, 
are  mentioned  here. 


192  THE   URINE 

Acetanilid  and  Phenacetin. — The  urine  is  evaporated 

by  gentle  heat  to  about  half  its  volume,  boiled  for  a  few 
minutes  with  about  one-fifth  its  volume  of  strong  hydro- 
chloric acid,  and  shaken  out  with  ether.  The  ether  is 
evaporated,  the  residue  dissolved  in  water,  and  the 
following  test  applied:  To  about  10  c.c.  are  added  a  few 
cubic  centimeters  of  3  per  cent,  phenol,  followed  by  a 
weak  solution  of  chromium  trioxid  (chromic  acid)  drop 
by  drop.  The  fluid  assumes  a  red  color,  which  changes 
to  blue  when  ammonia  is  added.  If  the  urine  is  very 
pale,  extraction  with  ether  may  be  omitted. 

Antipjrrin. — This  drug  gives  a  dark-red  color  when  a 
few  drops  of  10  per  cent,  ferric  chlorid  are  added  to  the 
urine.  The  color  does  not  disappear  up>on  boiling, 
which  excludes  diacetic  acid. 

Arsenic. — ReinscVs  Test. — Add  to  the  urine  in  a  test- 
tube  or  small  flask  about  one-seventh  its  volume  of  hy- 
drochloric acid,  introduce  a  piece  of  bright  copper-foil 
about  J-^  inch  square,  and  boil  for  several  minutes. 
If  arsenic  be  present,  a  dark-gray  film  is  deposited 
upon  the  copper.  The  test  is  more  delicate  if  the  urine 
be  concentrated  by  slow  evaporation.  This  test  is  well 
known  and  is  widely  used,  but  is  not  so  reliable  as  the 
following: 

GutzeWs  Test. — In  a  large  test-tube  place  a  little 
arsenic-free  zinc,  and  add  5  to  10  c.c.  pure  dilute  hydro- 
chloric acid  and  a  few  drops  of  iodin  solution  (Gram's 
solution  will  answer),  then  add  5  to  10  c.c.  of  the  urine. 
At  once  cover  the  mouth  of  the  tube  with  a  filter-paper 
cap  moistened  with  saturated  aqueous  solution  of  sOver 
nitrate  (1:1)  If  arsenic  be  present,  the  paper  quickly 
becomes  lemon  yellow,  owing  to  formation  of  a  coni- 


CHEMICAL   EXAMINATION  1 93 

pound  of  silver  arsenid  and  silver  nitrate,  and  turns 
black  when  touched  with  a  drop  of  water.  To  make 
sure  that  the  reagents  are  arsenic-free,  the  paper  cap 
may  be  applied  for  a  f«w  minutes  before  the  urine  is 
added. 

Atropin  will  cause  dilatation  of  the  pupil  when  a  few 
drops  of  the  urine  are  placed  in  the  eye  of  a  cat  or 
rabbit. 

Bromids  can  be  detected  by  acidifying  about  lo  c.c.  of 
the  urine  with  dilute  sulphuric  acid,  adding  a  few  drops 
of  fuming  nitric  acid  and  a  few  cubic  centimeters  of 
chloroform,  and  shaking.  In  the  presence  of  bromin  the 
chloroform,  which  settles  to  the  bottom,  assumes  a 
yellow  color. 

Chloral  hydrate  appears  in  the  urine  chiefly  as  uro- 
chloralic  acid,  which  reduces  the  copper  solutions  used 
for  sugar  tests.  To  detect  it,  evaporate  about  500  c.c. 
of  the  urine  to  about  one-fourth  its  volume,  make 
decidedly  add  with  hydrochloric  acid,  add  about  50  c.c. 
of  ether,  shake  thoroughly,  and  separate  the  ether. 
Now  evaporate  the  ether  and  dissolve  the  residue  in  a 
little  water.  If  urochloralic  acid  be  present  this 
aqueous  solution  will  respond  to  Fehling's  test. 

Hexamethylenamin. — Interest  in  this  drug  centers 
chiefly  in  its  value  as  a  urinary'  antiseptic,  which  de- 
pends upon  its  decomposition  with  liberation  of  for- 
maldehyd.  According  to  a  number  of  recent  workers 
formaldehyd  can  be  detected  in  the  urine  of  only  about 
50  per  cent,  of  patients  who  are  taking  hexamethylen- 
amin.  A  test  for  formaldehyd  is,  therefore,  necessar\' 
in  order  to  know  whether  the  object  in  administering 
the  drug  is  being  accomplished. 


194  THE   URINE 

Rimini-Bumam  Test  for  Formaldehyd. — To  about  lo  c.c. 
of  the  urine  add  successively  3  drops  of  0.5  per  cent,  solution 
of  phenylhydrazin  hydrochlorid,  3  drops  of  5  per  cent, 
solution  of  sodium  nitroprussid,  and  a  few  drops  of  a 
saturated  solution  of  sodium  hydroxid.  The  last  is  allowed 
to  trickle  down  the  inside  of  the  tube;  and  if  formaldehyd 
be  present  a  purplish-black  color,  changing  to  green  and 
then  to  yellow,  will  appear  as  it  mingles  with  the  urine. 

lodin  from  ingestion  of  iodids  or  absorption  from 
iodoform  dressings  is  tested  for  in  the  same  way  as  the 
bromids,  the  chloroform  assuming  a  pink  to  reddish- 
violet  color;  or  Obermayer's  reagent  may  be  used  in  the 
same  way  as  described  for  indican  (see  p.  134).  To  de- 
tect traces,  a  large  quantity  of  urine  should  be  rendered 
alkaline  with  sodium  carbonate  and  greatly  concen- 
trated by  evaporation  before  testing. 

Lead. — No  simple  method  is  sufficiently  sensitive  to 
detect  the  traces  of  lead  which  occur  in  the  urine  in 
chronic  poisoning.  Of  the  more  sensitive  methods,  that 
of  Arthur  Lederer  is  probably  best  suited  to  the  prac- 
titioner: 

It  is  essential  that  all  apparatus  used  be  lead-free. 
Five  hundred  cubic  centimeters  of  the  urine  are  acidified 
with  70  c.c.  pure  sulphuric  acid,  and  heated  in  a  beaker 
or  porcelain  dish.  About  20  to  25  Gm.  of  potassium 
persulphate  are  added  a  little  at  a  time.  This  should 
decolorize  the  urine,  leaving  it  only  slightly  yellow.  If 
it  darkens  upon  heating,  a  few  more  crystals  of  potas- 
sium persulphate  are  added,  the  burner  being  first  re- 
moved to  prevent  boiling  over;  if  it  becomes  cloudy,  a 
small  amount  of  sulphuric  acid  is  added.  It  is  then 
boiled  until  it  has  evaporated  to  250  c.c,  or  less.     After 


CHEMICAL   EXAMINATION  I95 

cooling,  an  equal  volume  of  alcohol  is  added,  and  the 
mixture  allowed  to  stand  in  a  cool  place  for  four  or  five 
hours,  during  whi'^h  time  all  the  lead  will  be  precipitated 
as  insoluble  sulphate. 

The  mixture  is  then  filtered  through  a  small,  close- 
grained  filter-paper  (preferably  an  ashless,  quantitative 
filter-paper) ,  and  any  sediment  remaining  in  the  beaker 
or  dish  is  carefully  washed  out  with  alcohol  and  filtered. 
A  test-tube  is  placed  underneath  the  funnel;  a  hole  is 


Fig.   48. — A  simple  hydrogen  sulphid  generator. 

punched  through  the  tip  of  the  filter  with  a  small  glass 
rod,  and  all  the  precipitate  (which  may  be  so  slight  as  to 
be  scarcely  visible)  washed  down  into  the  test-tube  with 
a  jet  of  distilled  water  from  a  wash-bottle,  using  as  little 
water  as  possible.  Ten  cubic  centimeters  will  usually 
suffice.  This  fluid  is  then  heated,  adding  crystals  of 
sodium  acetate  until  it  becomes  perfectly  clear.  It  now 
contains  all  the  lead  of  the  500  c.c.  urine  in  the  form  of 
lead  acetate.     It  is  allowed  to  cool,  and  hydrogen  sul- 


196  THE   URINE 

phid  gas  is  passed  through  it  for  about  five  minutes. 
The  slightest  yellowish-brown  discoloration  indicates 
the  presence  of  lead.  A  very  slight  discoloration  can  be 
best  seen  when  looked  at  from  above.  For  comparison, 
the  gas  may  be  passed  through  a  test-tube  containing 
an  equal  amount  of  distilled  water.  The  quantity  of 
lead  can  be  determined  by  comparing  the  discoloration 
with  that  produced  by  passing  the  gas  through  lead 
acetate  (sugar  of  lead)  solutions  of  known  strength. 
One  gram  of  lead  acetate  crystals  contains  0.54  Gm.  of 
lead.  Hydrogen  sulphid  is  easily  prepared  in  the  simple 
apparatus  shown  in  Fig.  48.  A  small  quantity  of  iron 
sulphid  is  placed  in  the  test-tube;  a  little  dilute  hydro- 
chloric acid  is  added;  the  cork  is  replaced;  and  the  deliv- 
ery tube  is  inserted  to  the  bottom  of  the  fluid  to  be 
tested. 

Mercury. — Traces  can  be  detected  in  the  urine  for  a 
considerable  time  after  the  use  of  mercury  compounds 
by  ingestion  or  inunction. 

About  a  liter  of  urine  is  acidified  with  10  c.c.  hydro- 
chloric acid,  and  a  small  piece  of  copper-foil  or  gauze  is 
introduced.  This  is  gently  heated  for  an  hour,  and 
allowed  to  stand  for  .twenty-four  hours.  The  metal  is 
then  removed,  and  washed  successively  with  very  dilute 
sodium  hydroxid  solution,  alcohol,  and  ether.  When 
dry,  it  is  placed  in  a  long,  slender  test-tube,  and  the 
lower  portion  of  the  tube  is  heated  to  redness.  A  tube 
with  a  constriction  in  its  upper  portion  is  better.  If 
mercury  be  present,  it  will  volatilize  and  condense  in 
the  upper  portion  of  the  tube  as  small,  shining  globules 
which  can  be  seen  with  a  hand-magnifier  or  low  power 
of  the  microscope.     If,  now,  a  crystal  of  iodin  be  drop)- 


CHEMICAL    EXAMTN'ATIOX  I97 

ped  into  the  tube  and  gently  heated,  the  mercurj-  upon 
the  side  of  the  tube  is  changed  nrst  to  the  yellow  iodid. 
and  later  to  the  red  iodid.  which  are  recx>gnized  by  their 
color. 

Morphin. — Add  sufficient  ammonia  to  the  urine  to 
render  it  distinctly  ammoniacal,  and  shake  thoroughly 
with  a  considerable  quantity  of  pure  acetic  ether. 
Separate  the  ether  and  evaporate  to  dryness.  To  a 
little  of  the  residue  in  a  watch-glass  or  porcelain  dish 
add  a  few  drops  of  formaldehyd-sulphuric  add,  which 
has  been  freshly  prepared  by  adding  i  drop  of  formalin 
to  I  c.c.  pure  concentrated  sulphuric  acid.  If  morphin 
be  present,  this  will  produce  a  purple-red  color,  which 
changes  to  ^•iolet.  blue\'iolet,  andfinally  nearly  pure  blue. 

PhenoL — As  has  been  stated,  the  urine  following 
phenol-poisoning  turns  olive  green  and  then  brownish 
black  up)on  standing.  Tests  are  of  value  in  recognizing 
poisoning  from  ingestion  and  in  detecting  absorption 
from  carbolized  dressings. 

The  urine  is  addulated  with  hydrochloric  acid  and 
distilled.  To  the  first  few  cubic  centimeters  of  distillate 
is  added  lo  per  cent,  solution  of  ferric  chlorid  drop  by 
drop.  The  presence  of  phenol  causes  a  deep  amethyst- 
blue  color,  as  in  Uffelmann's  test  for  lactic  add  (see 
p.  402). 

I^enolphthalein,  which  is  now  widely  used  as  a  ca- 
thartic, gives  a  bright  pink  color  when  the  urine  is  ren- 
dered alkaline. 

Quinin  — A  considerable  quantity  of  the  urine  is  roi- 
dered  alkaline  with  anmionia  and  extracted  with  ether; 
the  ether  is  evaporated,  and  a  portion  of  the  residue 
dissolved  in  about  20  drops  of  dilute  alcohol.     The 


198  THE    URINE 

alcoholic  solution  is  acidulated  with  dilute  sulphuric 
acid,  I  drop  of  an  alcoholic  solution  of  iodin  (tincture 
of  iodin  diluted  about  ten  times)  is  added,  and  the  mix- 
ture is  warmed.  Upon  cooling,  an  iodin  compound  of 
quinin  (herapathite)  will  separate  out  in  the  form  of  a 
microcrystalline  sediment  of  green  plates. 

The  remainder  of  the  residue  may  be  dissolved  in  a 
little  dilute  sulphuric  acid.  This  solution  will  show  a 
characteristic  blue  fluorescence  when  quinin  is  present. 

Resinous  drugs  cause  a  white  precipitate  like  that  of 
albumin  when  strong  nitric  acid  is  added  to  the  urine. 
This  is  dissolved  by  alcohol. 

Salicylates,  salol,  aspirin,  and  similar  drugs  give  a 
bluish- violet  color,  which  does  not  disappear  upon  heat- 
ing, upon  addition  of  a  few  drops  of  10  per  cent,  ferric 
chlorid  solution.  When  the  quantity  of  salicylates  is 
small,  the  urine  may  be  acidified  with  hydrochloric  acid 
and  extracted  with  ether,  the  ether  evaporated,  and  the 
test  applied  to  an  aqueous  solution  of  the  residue. 

Tannin  and  its  compounds  appear  in  the  urine  as 
gallic  acid,  and  the  urine  becomes  greenish  black  (inky, 
if  much  gallic  acid  be  present)  when  treated  with  a  solu- 
tion of  ferric  chlorid. 

IV.  MICROSCOPIC  EXAMINATION 

A  careful  microscopic  examination  will  often  reveal 
structures  of  great  diagnostic  importance  in  urine  which 
seems  perfectly  clear,  and  from  which  only  very  slight 
sediment  can  be  obtained  with  the  centrifuge.  Upon 
the  other  hand,  cloudy  urines  with  abundant  sediment 
are  often  shown  by  the  microscope  to  contain  nothing 
of  clinical  significance. 


MICROSCOPIC    EXAMINATION  1 99 

Since  the  nature  of  the  sediment  soon  changes,  the 
urine  must  be  examined  while  fresh,  preferably  within 
six  hours  after  it  is  voided.  When  possible  it  should  be 
kept  on  ice.-  The  sediment  is  best  obtained  by  means  of 
the  centrifuge.  If  a  centrifuge  is  not  available,  the 
urine  rfiay  be  allowed  to  stand  in  a  conical  test-glass  for 
six  to  twenty-four  hours  after  adding  some  preservative 
(see  p.  loo). 

A  small  amount  of  the  sediment  should  be  transferred 
to  a  slide  by  means  of  a  pipet.  It  is  very  important  to 
do  this  properly.  The  best  pipet  is  a  simple  glass  tube  7 
or  8  inches  long  which  has  been  drawn  out  at  one  end  to 
a  tip  with  a  I  or  1.5-mm.  opening.  The  centrifuge  tube 
containing  the  sediment  is  held  on  a  level  with  the  eye, 
the  larger  end  of  the  pipet  is  closed  with  the  index-finger, 
which  must  be  dry,  and  the  tip  is  carried  down  into  the 
sediment.  By  carefully  loosening  the  finger,  but  not 
entirely  removing  it,  a  small  amount  of  the  sediment  is 
then  allowed  to  run  slowly  into  the  pipet.  Slightly 
rotating  the  pipet  will  aid  in  accomplishing  this,  and 
at  the  same  time  will  serve  to  loosen  any  structures 
which  cling  to  the  bottom  of  the  tube.  After  wiping  off 
the  urine  which  adheres  to  the  outside,  a  drop  from 
the  pipet  is  placed  upon  a  clean  slide.  A  hair  is  then 
placed  in  the  drop  and  a  large  cover-glass  applied. 
The  correct  size  of  the  drop  can  be  learned  only  by  ex- 
perience. It  should  not  be  so  large  as  to  float  the  cover- 
glass  about,  nor  so  small  as  to  leave  unoccupied  space 
beneath  the  cover.  Many  workers  use  no  cover.  This 
offers  a  thicker  layer  and  larger  area  of  urine,  the  chance 
of  finding  scanty  structures  being  proportionately  in- 
creased.    It  has  the  disadvantage  that  any  jarring  of 


2CX)  THE    URINE 

the  room  (as  by  persons  walking  about)  sets  the  micro- 
scopic field  into  vibratory  motion  and  makes  it  impos- 
sible to  see  anything  clearly;  and,  since  it  does  not  allow 
satisfactory  use  of  high-power  objectives,  one  cannot 
examine  details  as  carefully  as  one  often  wishes  to  do. 
It  is  true  that  a  cover  can  be  applied  later,  but  any 
structure  which  one  has  found  with  the  low  power  and 
wishes  to  study  with  the  high  is  sure  to  be  lost  when  the 
cover  is  applied.  A  large  cover-glass  (about  22  mm. 
square)  with  a  hair  beneath  it  avoids  these  disadvan- 
tages, and  gives  enough  urine  to  find  any  structures 
which  are  present  in  sufficient  number  to  have  clinical 
significance,  provided  other  points  in  the  technic  have 
been  right.  It  is  best,  however,  to  examine  several 
drops;  and,  when  the  sediment  is  abundant,  drops  from 
the  upper  and  lower  portions  should  be  examined 
separately. 

In  examining  urinary  sediments  microscopically  no 
fault  is  so  common,  nor  so  fatal  to  good  results,  as  im- 
proper illumination  (see  Fig.  6),  and  none  is  so  easily 
corrected.  The  light  should  be  central  and  very  sub- 
dued for  ordinary  work,  but  oblique  illumination,  ob- 
tained by  swinging  the  mirror  a  little  out  of  the  optical 
axis,  will  be  found  helpful  in  identifying  certain  delicate 
structures  like  hyaline  casts.  The  i6-mm.  objective 
should  be  used  as  a  finder,  while  the  4-mm.  is  reserved 
for  examining  details.  An  experienced  worker  will  rely 
almost  wholly  upon  the  lower  power. 

It  is  well  to  emphasize  that  the  most  common  errors 
which  result  in  failure  to  find  important  structures,  when 
present,  are:  (a)  lack  of  care  in  transferring  the  sediment  to 


MICROSCOPIC   EXAMINATION  201 

the  slide,  (b)  too  strong  illumination,  and  (c)  too  great 
magnification. 

In  order  to  distinguit^h  between  similar  structures  it  is 
often  necessary  to  watch  the  effect  upon  them  of  certain 
reagents.  This  is  especially  true  of  the  various  un- 
organized sediments.  They  very  frequently  cannot  be 
identified  from  their  form  alone.  With  the  structures 
still  in  focus,  a  drop  of  the  reagent  may  be  placed  at  one 
edge  of  the  cover-glass  and  drawn  underneath  it  by 
the  suction  of  a  piece  of  blotting-paper  touched  to  the 
opposite  edge;  or,  better,  a  small  drop  of  the  reagent 
and  of  the  urine  may  be  placed  close  together  upon  a 
slide  and  a  cover  gently  lowered  over  them.  As  the 
two  fluids  mingle,  the  effect  upon  various  structures 
may  be  seen. 

Urinary  sediments  may  be  studied  under  three  heads : 
A.  Unorganized  sediments.  B.  Organized  sediments. 
C.  Extraneous  structures. 

A.    Unorganized  Sediments 

In  general,  these  have  little  diagnostic  or  prognostic 
significance.  Most  of  them  are  substances  normally 
present  in  solution,  which  have  been  precipitated  either 
because  present  in  excessive  amounts,  or,  more  fre- 
quently, because  of  some  alteration  in  the  urine  (as  in 
reaction,  concentration,  etc.)  which  may  be  purely 
physiologic,  depending  upon  changes  in  diet  or  habits. 
Various  substances  are  always  precipitated  during  de- 
composition, which  may  take  place  either  within  or 
without  the  body.  Unorganized  sediments  may  be 
classified  according  to  the  reaction  of  the  urine  in  which 


202 


THE    URINE 


they  are  most  likely  to  be  found.  This  classification  is 
usetul,  but  is  not  accurate,  since  the  characteristic  sedi- 
ments of  acid  urine  may  remain  after  the  urine  has 
become  alkaline,  while  the  alkaline  sediments  may  be 
precipitated  in  a  urine  which  is  still  acid. 

In  acid  urine:  Uric  acid,  amorphous  urates,  sodium 
urate,  calcium  oxalate,  leucin  and  tyrosin,  cystin,  and 
fat-globules.     Uric  acid,  the  urates,  and  calcium  oxalate 


Fig.  49. — Unusual  urinary  crystals  (drawn  from  vari.  ;.  rs): 

I,  Calcium  sulphate  (colorless);  2,  cholesterol  (colorless);  3,  hippuric 
acid  (colorless);  4,  hematoidin  (brown);  5,  fatty  acids  (colorless);  6, 
indigo  (blue);  7,  sodium  urate  (yellowish). 


are  the  common  deposits  of  acid  urines;  the  others  are 
less  frequent,  and  depend  less  upon  the  reaction  of  the 
urine. 

In  alkaline  urine:  Phosphates,  calcium  carbonate,  and 
ammonium  urate. 

Other  crystalline  sediments  (Fig.  49)  which  are  rare 
and  require  no  further  mention  are:  Calcium  sulphate, 
cholesterol,  hippuric  acid,  hematoidin,  fatty  acids,  and 
indigo. 

The  following  brief  table  will  aid  the  student  in  identi- 
fying the  chemical  sediments  which  one  meets  every  day: 


PLATE   III. 


Fig.  I. — Common  sediments  of  alkaline  urine:  Triple  phosphate 
crystals,  calcium  phosphate  crystals,  ammonium  urate  crystals,  and 
amorphous  phosphates.     X  150. 


"T-^-^ 


^^. 


)£m 


a 


t-  '^ 


Fig.  2. — Cormnon  sediments  of  acid  urine:    Uric-acid  crystals,  calcium 
oxalate  crystals,  and  amorphous  urates.     X  150. 


MICROSCOPIC    EXAMINATION 


203 


In  acid  urine 

In  alkaline  urine 

Yellow  crystals. 

Uric  acid — dissolve  in 
KOH. 

Ammonium  urate — dis- 
solve in  HCl. 

Colorless  crystals. 

Calcium  oxalate— dis- 
solve in  HCl. 

Phosphate  crystals — 
dissolve  in  acetic  acid. 

Amorphous  material. 

Urates — dissolve    with 
heat. 

Amorphous  phosphates 
— dissolve  in  acetic 
acid. 

1.  In  Acid  Urine. — (i)  Uric -acid  Crystals. — These 
crystals  are  the  red  grains — "gravel"  or  "red  sand" 
— which  are  often  seen  adhering  to  the  sides  and 
bottom  of  a  vessel  containing  urine.  Microscopically, 
they  are  yellow  or  reddish-brown  crystals,  which  differ 
greatly  in  size  and  shape.  The  color  is  due  to  urinary 
pigments,  chiefly  uroerythrin.  The  most  characteristic 
forms  (Plate  III  and  Fig.  50)  are  "whetstones;" 
roset-like  clusters  of  prisms  and  whetstones;  and  rhom- 
bic plates,  which  have  usually  a  paler  color  than  the 
other  forms  and  are  sometimes  colorless.  A  very 
rare  form  is  a  colorless  hexagonal  plate  resembling 
cystin.  Recognition  of  the  crystals  depends  less 
upon  their  shape  than  upon  their  color,  the  reaction  of 
the  urine,  and  the  facts  that  they  are  soluble  in  caustic 
soda  solution  and  insoluble  in  hydrochloric  or  acetic 
acid.  When  ammonia  is  added,  they  dissolve  and 
crystals  of  ammonium  urate  appear. 

A  deposit  of  uric-acid  crystals  has  no  significance  un- 
less it  occurs  before  or  very  soon  after  the  urine  is  voided. 
Every  urine,  if  kept  acid,  will  in  time  deposit  its  uric 


204 


THE    URINE 


acid.  Factors  which  favor  an  early  deposit  are  high 
acidity,  diminished  urinary  pigments,  and  excessive  ex- 
cretion of  uric  acid.  The  chief  clinical  interest  of  the 
crystals  lies  in  their  tendency  to  form  calculi,  owing  to 
the  readiness  with  which  they  collect  about  any  solid 
object.     Their  presence  in  the  freshly  voided  urine  in 


Fig.  so. — Forms  of  uric  acid:  i,  Rhombic  plates;  2,  whetstone 
forms;  3,  3,  quadrate  forms;  4,  5,  prolonged  into  points;  6,  8,  resets; 
7,  pointed  bundles;  8,  barrel  forms  precipitated  by  adding  hydrochloric 
acid  to  urine  (Ogden). 

clusters  of  crystals  suggests  stone  in  the  kidney  or  blad- 
der, especially  if  blood  is  also  present  (see  Fig.  82). 

It  was  formerly  believed  that  the  uric  acid  stone  is 
the  most  common  form  of  renal  calculus,  but  from  a 
recent  study  of  a  series  of  calculi  Kahn  and  Rosen- 
bloom  believe  that  the  great  majority  are  composed  of 
calcium  oxalate  although  all  contain  a  trace  of  uric  acid. 


MICROSCOPIC   EXAMINATION  205 

(2)  Amorphous  Urates. — These  are  chiefly  urates  of 
sodium  and  potassium  which  are  thrown  out  of  solution 
as  a  yellow  or  red  "brick-dust "  deposit.  In  pale  urines 
this  sediment  is  almost  white.  It  disappears  upon 
heating.  A  deposit  of  amorphous  urates  is  very  com- 
mon in  concentrated  and  strongly  acid  urines,  especially 
in  cold  weather,  and  has  no  clinical  significance.  Under 
the  microscope  it  appears  as  fine  yellowish  granules, 
sometimes  almost  colorless  (Plate  III).  Often  they 
are  so  abundant  as  to  obscure  all  other  structures. 
In  such  cases  the  urine  should  be  warmed  before 
examining.  The  granules  have  a  tendency  to  collect 
upon  tube-casts,  strands  of  mucus,  and  other  structures. 
Amorphous  urates  are  readily  soluble  in  caustic  soda 
solutions.  When  treated  with  hydrochloric  or  acetic 
acid,  they  slowly  dissolve  and  rhombic  crystals  of 
uric  acid  appear  in  ten  to  twenty  minutes. 

Rarely,  sodium  urate  occurs  in  crystalline  form — 
slender  prisms,  arranged  in  fan-  or  sheaf-like  structures 
(see  Fig.  49). 

(3)  Calcium  Oxalate. — Characteristic  of  calcium  oxa- 
late are  colorless,  glistening,  octahedral  crystals,  giving 
the  appearance  of  small  squares  crossed  by  two  intersect- 
ing diagonal  lines — the  so-called  "envelope  crystals" 
(see  Fig.  77).  They  vary  greatly  in  size,  being  some- 
times so  small  as  to  seem  mere  points  of  light  with 
medium-power  objectives.  Unusual  forms,  which, 
however,  seldom  occur  except  in  conjunction  with  the 
octahedra,  are  colorless  dumb-bells,  spheres,  and  varia- 
tions of  the  octahedra  (Fig.  51).  The  spheres  might 
be  mistaken  for  globules  of  fat  or  red  blood-corpuscles. 
Crystals  of  calcium  oxalate  are  insoluble  in  acetic  acid 


2o6  THE   URINE 

or  caustic  soda.  They  are  dissolved  by  strong  hydro- 
chloric acid,  and  recrystallize  as  octahedra  upon  addi- 
tion of  ammonia.  They  are  sometimes  encountered  in 
alkaline  urine. 

The  crystals  are  commonly  found  in  the  urine  after 
ingestion  of  vegetables  rich  in  oxalic  acid,  as  tomatoes, 


Fig.  51. — Various  forms  of  calcium  oxalate  crystals  from  urine. 
The  majority  are  the  typical  octahedra  seen  in  different  positions 
(X  450). 


spinach,  asparagus,  and  rhubarb.  They  have  no  defi- 
nite significance  pathologically.  They  often  appear  in 
digestive  disturbances,  in  neurasthenia,  and  when  the 
oxidizing  power  of  the  system  is  diminished.  When 
abundant,  they  are  generally  associated  with  a  little 
mucus;  and,  in  men,  frequently  with  a  few  spermatozoa. 
Their  chief  clinical  interest  lies  in  their  tendency  to 
form  calculi,  and  their  presence  in  fresh  urine,  together 
with  evidences  of  renal  or  cystic  irritation,  should  be 


MICROSCOPIC    EXAMINATION  207 

viewed  with  suspicion,  particularly  if  they  are  clumped 
in  small  masses. 

(4)  Leucin  and  Tyrosin. — These  substances  are  cleav- 
age products  of  the  protein  molecule.  They  are  of  com- 
paratively rare  occurrence  in  the  urine  and  generally 
appear  together.  In  general,  their  presence  indicates 
autolysis  of  tissue  proteins.  Clinically,  they  are  seen 
most  frequently  in  severe  fatty  destruction  of  the  liver, 
such  as  occurs  in  acute  yellow  atrophy  and  phosphorus- 
poisoning.  Crystals  are  deposited  spontaneously  only 
when  the  substances  are  present  in  large  amount.  Usu- 
ally they  will  be  deposited  when  the  urine  is  evaporated 
to  a  small  volume  on  a  water-bath.  It  is  best,  however, 
to  separate  them  from  the  urine  as  follows: 

Treat  500  to  1000  c.c.  of  urine,  which  has  been  freed  from 
albumin,  with  neutral,  then  with  basic,  lead  acetate  until  a 
precipitate  no  longer  forms.  Filter,  remove  excess  of  lead 
with  hydrogen  sulphid  (see  p.  196),  and  filter  again.  Con- 
centrate to  a  syrup  on  a  water-bath.  Extract  repeatedly 
with  small  quantities  of  absolute  alcohol  to  remove  urea. 
Treat  the  residue  with  hot  dilute  alcohol  to  which  a  little 
ammonia  has  been  added.  Filter  and  evaporate  the  filtrate 
to  a  small  volume  and  let  stand  for  the  leucin  and  tyrosin  to 
separate  out.  The  leucin  can  be  separated  from  the  tyrosin 
by  boiling  with  glacial  acetic  acid.  Leucin  dissolves,  leaving 
the  tyrosin,  and  can  again  be  recovered  by  evaporating  the 
acetic  acid. 

The  crystals  cannot  be  identified  from  their  mor- 
phology alone,  since  other  substances,  notably  calcium 
phosphate  (see  Fig.  57)  and  ammonium  urate,  may  take 
similar  or  identical  forms.     It  is,  therefore,  necessary  to 


2o8  THE    URINE 

try  out  their  solubility  in  various  reagents  or  to  apply 
special  tests. 

Leucin  crystals  (Fig.  52)  as  they  appear  in  the  urine 
do  not  represent  the  pure  substance.  They  are  slightly 
yellow,  oily-looking  spheres,  many  of  them  with  radial 
and  concentric  striations.  Some  may  be  merged  to- 
gether in  clusters.  They  are  not  soluble  in  hydrochloric 
acid  nor  in  ether. 


Fig.  52. — Leucin  spheres  and  tyrosin  needles  (Stengel). 

Tyrosin  crystallizes  in  very  fine  needles,  which  may 
appear  black  and  which  are  usually  arranged  in  sheaves, 
with  a  marked  constriction  at  the  middle  (Fig.  53).  It 
is  soluble  in  ammonia  and  hydrochloric  acid,  but  not  in 
acetic  acid. 

Momer's  Test  for  Tyrosin. — To  a  small  quantity  of  the 
crystals  in  a  test-tube  add  a  few  cubic  centimeters  of 
Momer's  reagent  (formalin,  i  c.c;  distilled  water,  45  c.c; 
concentrated  sulphuric  acid,  55  c.c).  Heat  gently  to  the 
boiling-point.     A  green  color  shows  the  presence  of  tyrosin. 

(5)  Cystin  crystals  are  colorless,  highly  refractive, 
rather  thick,  hexagonal  plates  with  well-defined  edges. 


MICROSCOPIC    EXAMINATION  209 

They  lie  either  singly  or  superimposed  to  form  more  or 
less  irregular  clusters  (Fig.  54).  Uric  acid  sometimes 
takes  this  form  and  must  be  excluded.  Cystin  is  soluble 
in  hydrochloric  acid,  insoluble  in  acetic;  it  is  readily 
soluble  in  ammonia  and  recrystallizes  upon  addition  of 
acetic  acid. 


Imc;.   53. — Tyrosin  crystals  from  urine  (  X  450). 

Cystin  is  one  of  the  amino-acids  formed  in  decompo- 
sition of  the  protein  molecule,  and  is  present  in  traces  in 
normal  urine.  Crystals  are  deposited  only  when  the 
substance  is  present  in  excessive  amount.  Their 
presence  is  known  as  cystinuria.  It  is  a  rare  condition 
due  to  an  obscure  abnormality  of  protein  metabolism 
and  usually  continues  throughout  life.  The  amount 
of  cystin  can  be  greatly  diminished  by  a  low-protein 
diet,  and  the  formation  of  crystals  can  in  some  measure 
be  prevented  by  administration  of  sodium  carbonate. 
There  are  rarely  any  symptoms  save  those  referable 

14 


2IO 


THE   URINE 


to  renal  or  cystic   calculus,   to  which   the   condition 
strongly  predisposes. 

(6)  Fat-globules. — Fat  appears  in  the  urine  as  highly 
refractive  globules  of  various  sizes,  frequently  very 
small.  These  globules  are  easily  recognized  from  the 
fact  that  they  are  stained  black  by  osmic  acid  and 
orange  or  red  by  Sudan  III.     The  stain  may  be  applied 


Fig.  54. — Cystin  crystals  from  urine  of  patient  with  cystin  calculus 

(  X  200). 


upon  the  slide,  as  already  described  (see  p.  201). 
Osmic  acid  should  be  used  in  i  per  cent,  aqueous  solu- 
tion; Sudan  III,  in  saturated  solution  in  70  per  cent, 
alcohol,  to  which  one-half  its  volume  of  10  per  cent, 
formalin  may  advantageously  be  added. 

Fat  in  the  urine  is  usually  a  contamination  from  un- 
clean vessels,  oiled  catheters,  etc.  A  very  small  amount 
may  be  present  after  ingestion  of  large  quantities  of  cod- 
liver  oil  or  other  fats.     In  fatty  degeneration  of   the 


MICROSCOPIC    EXAMINATION  211 

kidney,  as  in  phosphorus-poisoning  and  chronic  paren- 
chymatous nephritis,  fat-globules  are  commonly  seen, 
both  free  in  the  urine  and  embedded  in  cells  and  tube- 
casts.  Fat-droplets  are  common  in  pus-corpuscles  and 
in  degenerating  cells  of  any  kind. 

In  chyluria,  or  admixture  of  chyle  with  the  urine  as  a 
result  of  rupture  of  a  lymph-vessel,  minute  droplets  of 
fat  are  so  numerous  as  to  give  the  urine  a  milky  appear- 
ance. The  droplets  are  smaller  than  those  of  milk, 
which  is  sometimes  added  by  malingerers.  The  fluid 
is  often  blood-tinged.  The  condition  is  best  recognized 
by  shaking  up  with  ether,  which,  when  separated,  leaves 
the  urine  comparatively  clear.  If,  then,  the  ether  be 
evaporated  a  fatty  residue  remains.  Chyluria  occurs 
most  frequently  as  a  symptom  of  infection  by  filaria 
(see  p.  462),  the  larvae  of  which  can  usually  be 
found  in  the  milky  urine.  In  other  cases  the  etiology 
is  obscure. 

2.  In  Alkaline  Urine. — (i)  Phosphates. — While 
most  common  in  alkaline  urine,  phosphates  are  some- 
times deposited  in  amphoteric  or  feebly  acid  urines. 
The  usual  forms  are:  (a)  Ammoniomagnesium  phos- 
phate crystals;  (b)  acid  calcium  phosphate  crystals, 
and  (c)  amorphous  phosphates.  All  are  readily  soluble 
in  acetic  acid. 

(a)  Ammoniomagnesium  Phosphate  Crystals. — They  "^ 
are  the  common  "triple  phosphate"  crystals,  which  are  ^ 
generally  easily  recognized  (Figs.  55,  56  and  83,  and 
Plate  III) .  They  are  colorless,  except  when  bile  stained. 
Their  usual  form  is  some  modification  of  the  prism, 
with  oblique  ends.  Most  typical  are  the  well-known 
"coffin-lid"    and    "hip-roof"    forms.     The  long  axis 


212 


THE   URTNE 


Fig.  55. — Prismatic  forms  of  triple   phosphate   crystals,  from  urine 

(  X  450). 


Fig,  56. — Triple  phosphate  crystals:  forms  produced  by  rapid  precipi- 
tation and  by  partial  solution  of  prisms  (  X  450). 


MICROSCOPIC   EXAMINATION  2I3 

of  the  hip-roof  cr>-stal  is  often  so  shortened  that  it 
resembles  the  envelope  crystal  of  calcium  oxalate. 
It  does  not,  however,  have  the  same  luster;  this,  and 
its  solubility  in  acetic  acid,  will  alwa\-s  prevent 
confusion. 

When  rapidly  deposited,  as  by  artificial  precipitation, 
Jriple  phosphate  often  takes  feathery,  star-,  or  leaf-like 
forms  (Fig.  56).  These  gradually  develop  into  the  more 
common  prisms.  X-forms  may  be  prodiiced  by  partial 
solution  of  prisms. 


4 


? ; :    5  -  —  _  r  :alcmin  i^xisphate:  i.  Cor. 

from    Rieder's   AtlasJ ;    2.    needles  resembling   tyrosia    ;dravm    trom 
nature);  3.  large,  irregnlar  {dates  (from  natore). 

(6)  Dicalcium  Phosphate  Crystals. — In  feebly  add, 
amphoteric,  or  feebly  alkaline  urines  add  caldum  pho&- 
phate,  wrongly  called  "neutral  caldum  phosphate," 
is  not  infrequently  deposited  in  the  form  of  colorless 
prisms  arranged  in  stars  and  rosets  (Fig.  57,  i).  Be- 
cause of  the  shape  of  the  crystals  it  is  sometimes  called 
"  stellar  phosphate."  The  indiddual  prisms  are  usually 
slender,  with  one  beveled,  wedge-like  end,  but  are  some- 
times needle-like.  They  may  sometimes  take  forms 
resembling  tyrosin  (Fig.  57,  2),  caldum  sulphate,  ot 


214  THE   URINE 

hippuric  acid,  but  are  readily  distinguished  by  their 
solubility  in  acetic  acid. 

Calcium  phosphate  often  forms  large,  thin,  irregular, 
usually  granular,  colorless  plates  (Fig.  57,  3)  which 
should  be  easily  recognized,  although  small  plates 
might  be  mistaken  for  squamous  epithelial  cells.  These 
crystals  most  frequently  form  a  scum  upon  the  surface 
of  the  urine.  They  are  regarded  by  some  as  magnesium 
phosphate. 

Fig.  58. — Indistinct  crystalline  sediment  (dumb-bell  crystals)  of 
calcium  carbonate.  Similar  crystals  are  sometimes  formed  by  calcium 
oxalate  and  calcium  sulphate  (after  Funke). 

(c)  Amorphous  Phosphates. — The  earthy  phosphates 
are  thrown  out  of  solution  in  most  alkaline  and  many 
amphoteric  urines  as  a  white,  amorphous  sediment, 
which  may  be  mistaken  for  pus  macroscopically. 
Under  the  microscope  the  sediment  is  seen  to  consist 
of  numerous  colorless  granules,  distinguished  from 
amorphous  urates  by  their  color,  their  solubility  in 
acetic  acid,  and  the  reaction  of  the  urine. 

The  various  phosphatic  deposits  frequently  occur 
together.  They  are  sometimes  due  to  excessive  excre- 
tion of  phosphoric  acid,  but  usually  merely  indicate  that 
the  urine  has  become,  or  is  becoming,  alkaline  (see 
Phosphates,  p.  129). 

(2)  Calcium  Carbonate  may  sometimes  be  mingled 
with  the  phosphatic  deposits,  usually  as  amorphous 


MICROSCOPIC   EXAMINATION  215 

granules,  or,  more  rarely,  as  colorless  spheres  and  dumb- 
bells (Fig.  58),  which  are  soluble  in  acetic  acid  with  gas 
formation. 

(3)  Ammonium  Urate  Crystals. — This  is  the  only 
urate  deposited  in  alkaline  urine.  It  forms  opaque 
yellow  crystals,  usually  in  the  form  of  spheres  (Plate  III, 
and  Fig.  83),  which  are  often  covered  with  fine  or  coarse 


Fig.   59. — Crystals  of  ammonium  urate  (one-half  of  the  forms  copied 
from  Rieder's  Atlas;  the  others,  from  nature). 

spicules —  "thorn-apple  crystals."  Sometimes  dumb- 
bells, compact  sheaves  of  line  needles,  and  irregular 
rhizome  forms  are  seen  (Fig.  59) .  Upon  addition  of 
acetic  acid  they  dissolve,  and  rhombic  plates  of  uric 
acid  appear. 

These  crystals  occur  only  when  free  ammonia  is 
present.  They  are  generally  found  along  with  the  phos- 
phates in  decomposing  urine  and  have  no  clinical 
significance. 

B.  Organized  Sediments 

The  principal  organized  structures  in  urinary  sedi- 
ments are:  Tube-casts;  epithelial  cells;  pus-corpuscles; 


2l6  THE    URINE 

red  blood-corpuscles;  spermatozoa;  bacteria,  and  animal 
parasites.  They  are  much  more  important  than  the 
unorganized  sediments  just  considered. 

I.  Tube=casts. — These  interesting  structures  are 
albuminous  casts  of  the  uriniferous  tubules.  Their 
presence  in  the  urine  (known  as  cylindruria)  probably 
always  indicates  some  pathologic  change  in  the  kidney, 
although  this  change  may  be  very  slight  or  transitory. 
Large  numbers  may  be  present  in  temporary  irritations 
and  congestions.  They  do  not  in  themselves,  therefore, 
imply  organic  disease  of  the  kidney.  They  rarely  occur 
in  urine  whicl^  does  not  contain,  or  has  not  recently 
contained,  albumin. 

While  it  is  not  possible  to  draw  a  sharp  dividing  line 
between  the  different  varieties,  casts  may  be  classified 
as  follows: 

(i)  Hyaline  casts. 
(a)  Narrow. 
[h)  Broad. 

(2)  Waxy  casts. 

(3)  Fibrinous  casts. 

(4)  Granular  casts. 

(a)  Finely  granular. 
(&)  Coarsely  granular. 

(5)  Fatty  casts. 

(6)  Casts  containing  organized  structures. 
{a)  Epithelial  casts. 

{h)  Blood-casts. 
{c)  Pus-casts. 
{d)  Bacterial  casts. 
As  will  be  seen  later,  practically  all  varieties  are 
modifications  of  the  hyaline. 


MICROSCOPIC    EXAMINATION  21 7 

The  significance  of  the  different  varieties  is  more 
readily  understood  if  one  considers  their  mode  of  forma- 
tion. Albuminous  material,  the  source  and  nature  of 
which  are  not  definitely  known,  but  which  are  doubtless 
not  the  same  in  all  cases,  probably  enters  the  lumen  of  a 
uriniferous  tubule  in  a  fluid  or  plastic  state.  The  ma- 
terial has  been  variously  thought  to  be  an  exudate 
from  the  blood,  a  pathologic  secretion  of  the  renal  cells, 
and  a  product  of  epithelial  degeneration.  In  the  tubule 
it  hardens  into  a  cast  which,  when  washed  out  by  the 
urine,  retains  the  shape  of  the  tubule,  and  contains 
within  its  substance  whatever  structures  and  debris  were 
lying  free  within  the  tubule  or  were  loosely  attached  to 
its  wall.  If  the  tubule  be  small  and  has  its  usual  lining 
of  epithelium,  the  cast  will  be  narrow;  if  it  be  large  or 
entirely  denuded  of  epithelium,  the  cast  will  be  broad. 
A  cast,  therefore,  indicates  the  condition  of  the  tubule  in 
which  it  is  formed,  but  does  not  necessarily  indicate  the  con- 
dition of  the  kidney  as  a  whole.  In  any  particular  case 
of  kidney  disease  several  forms  or  even  all  may  be  found. 
Their  number  and  the  preponderance  of  certain  forms 
will,  as  is  shown  later,  furnish  a  clue  to  the  nature  of  the 
pathologic  process  but  further  than  this  one  cannot  go 
with  certainty.  One  cannot  rely  upon  the  casts  for 
accurate  diagnosis  of  the  histologic  changes  in  the 
kidney. 

At  times  during  the  course  of  a  nephritis  the 
urine  is  suddenly  flooded  with  great  numbers  of  tube- 
casts.  Such  "showers"  may  be  of  serious  import  but 
are  not  necessarily  so.  In  some  cases  they  may  result 
from  a  clearing  out  of  the  plugged  renal  tubules  coinci- 
dent with  improvement  and  increased  flow  of  urine. 


2l8  THE    URINE 

The  search  for  casts  must  be  carefully  made.  The 
urine  must  be  fresh,  since  hyaline  casts  soon  dissolve, 
when  it  becomes  alkaline.  It  should  be  thoroughly 
centrifugalized.  When  the  sediment  is  abundant,  casts, 
being  light  structures,  will  be  found  near  the  top  of  the 
sediment.  In  cystitis,  where  casts  may  be  entirely 
hidden  by  the  pus,  the  bladder  should  be  irrigated  to 
remove  as  much  of  the  pus  as  possible  and  the  next  urine 
examined.  In  order  to  prevent  solution  of  the  casts  the 
urine,  if  alkaline,  must  be  rendered  acid  by  previous 
administration  of  boric  acid  or  other  drugs.  Heavy 
sediments  of  urates,  blood,  or  vaginal  cells  may  like- 
wise obscure  casts  and  other  important  structures.  The 
last  can  be  avoided  by  catheterization.  Urates  can  be 
dissolved  by  gently  warming  before  centrifugalizing, 
care  being  taken  not  to  heat  enough  to  coagulate  the 
albumin.  The  aluminum  shield  of  the  centrifuge  tube 
may  also  be  heated.  Blood  can  be  destroyed  by  centri- 
fugalizing, pouring  off  the  supernatant  urine,  filling  the 
tube  with  water,  adding  a  few  drops  of  dilute  acetic  acid, 
mixing  well,  and  again  centrifugalizing;  this  process 
being  repeated  until  the  blood  is  completely  decolorized. 
Too  much  acetic  acid  will  dissolve  hyaline  casts. 

In  searching  for  casts  the  low-power  objective  should 
invariably  be  used,  although  a  higher  power  may  occa- 
sionally be  desirable  in  studying  details  as,  for  example, 
in  distinguishing  between  an  epithelial  and  a  pus-cast. 
The  casts  are  perhaps  most  frequently  found  near  the 
edge  of  the  cover-glass.  Their  cylindric  shape  can  be 
best  seen  by  slightly  moving  the  cover-glass  while  ob- 
serving them,  or  by  pressing  upon  one  edge  of  the 
cover  with  a  needle,  thus  causing  them  to  roll.     This 


MICROSCOPIC   EXAMINATION  219 

little  manipulation  should  be  practised  until  it  can  be 
done  satisfactorily.  It  will  prove  useful  in  many 
examinations. 

Various  methods  of  staining  casts  so  as  to  render  them 
more  conspicuous  have  been  proposed.  They  offer  no 
special  advantage  to  one  who  understands  how  to  use 
the  substage  mechanism  of  his  microscope.  The  "  nega- 
tive-staining" method  is  as  good  as  any.  It  consists 
simply  in  adding  a  little  India-ink  to  the  drop  of  urine 
on  the  slide.  Casts,  cells,  etc.,  will  stand  out  as  colorless 
structures  on  a  dark  background. 

(i)  Hyaline  Casts.^Typically,  these  are  colorless, 
homogeneous,    semitransparent,    cylindric    structures, 


Fig.  60. — Hyaline  casts  showing  fat-droplets  and  leukocytes  (obj.  4 
mm.)  (Boston). 

with  parallel  sides  and  usually  rounded  ends.  Not  in- 
frequently they  are  more  opaque  or  show  a  few  granules 
or  an-  occasional  cell  or  oil-globule,  either  adhering  to 
them  or  contained  within  their  substance.  Generally 
they  are  straight  or  curved;  less  commonly,  convoluted. 


220 


THE   URINE 


Their  length  and  breadth  vary  greatly:  they  are  some- 
times so  long  as  to  extend  across  several  fields  of  a 
medium-power  objective,  but  are  usually  much  shorter; 
in  breadth  they  vary  from  one  to  seven  or  eight  times 
the  diameter  of  a  red  blood-corpuscle  (see  Figs.  6,  60, 
61,  and  66). 


Fig.  61. — Various  kinds  of  casts:  a.  Hyaline  and  finely  granular 
cast;  b,  finely  granular  cast;  c,  coarsely  granular  cast;  d,  brown  granu- 
lar cast;  e,  granular  cast  with  normal  and  abnormal  blood  cells  ad- 
herent; /,  granular  cast  with  renal  cells  adherent;  g,  granular  cast 
with  fat  and  a  fatty  renal  cell  adherent  (Ogden). 

Hyaline  casts  are  the  least  significant  of  all  the  casts, 
and  occur  in  many  slight  and  transitory  conditions. 
Small  numbers  are  common  following  ether  anesthesia, 
in  fevers,  after  excessive  exercise,  and  in  congestions  and 
irritations  of  the  kidney.  They  are  always  present,  and 
are  usually  stained  yellow  when  the  urine  contains  much 
bile.  While  they  are  found  in  all  organic  diseases  of  the 
kidney,  they  are  most  important  in  chronic  interstitial 
nephritis.  Here  they  are  seldom  abundant,  but  their 
constant  presence  is  the  most  reliable  urinary  sign  of  the 
disease.  Small  areas  of  chronic  interstitial  change  are 
probably  responsible  for  the  few  hyaline  casts  so  fre- 
quently found  in  the  urine  of  elderly  persons. 


MICROSCOPIC    EXAMINATION-  221 

Very  broad  hyaline  casts  commonly  indicate  complete 
desquamation  of  the  tubular  epithelium,  such  as  occurs 
in  the  late  stages  of  nephritis. 

(2)  "Waxy  Casts. — Like  hyaline  casts,  these  are  homo- 
geneous when  typical,  but  frequently  contain  a  few 
granules  or  an  occasional  cell.  They  are  much  more 
opaque  than  the  hyaline  variety,  and  are  usually  shorter 
and  broader,  with  irregular,  broken  ends,  and  some- 
times appear  to-  be  segmented.  They  are  grayish  or 
colorless,  and  have  a  dull,  waxy  look,  as  if  cut  from  par- 
affin (Figs.  62  and  8i).     They  are  sometimes  composed 


Fig.  62. — Waxy  casts  (upper  part  of  figure).     Fatty  and  fat-bearing 
casts  (lower  part  of  figure)  (from  Greene's  "  Medical  Diagnosis"). 

of  material  which  gives  the  amyloid  reactions.  All 
gradations  between  hyaline  and  waxy  casts  may  be 
found.  Waxy  casts  are  found  in  most  advanced  cases  of 
nephritis,  where  they  are  an  unfavorable  sign.  They 
are  perhaps  most  abundant  in  amyloid  disease  of  the 
kidney,  but  are  not  distinctive  of  the  disease,  as  is 
sometimes  stated. 

(3)  Fibrinous  Casts. — Casts  which  resemble  waxy 
casts,  but  have  a  distinctly  yellow  color,  as  if  cut  from 
beeswax,  are  often  seen  in  acute  nephritis.     They  are 


222 


THE   URINE 


called  fibrinous  casts,  but  the  name  is  inappropriate,  as 
they  are  not  composed  of  fibrin.  They  are  often  classed 
with  waxy  casts,  but  should  be  distinguished,  as  their 
significance  is  much  less  serious. 

(4)  Granular  Casts. — These  are  merely  hyaline  casts 
in  which  numerous  granules  are  embedded  (Figs.  61,  63, 
and  66). 

Finely  granular  casts  contain  many  fine  granules,  are 
usually  shorter,  broader,  and  more  opaque  than  the 
hyaline  variety,  and  are  more  conspicuous.  Their 
color  is  grayish  or  pale  yellow. 


Fig.  63. — Granular  and  fatty  casts  and  two  compound  granule  cells 

(Stengel). 

Coarsely  granular  casts  contain  larger  granules  and  are 
darker  in  color  than  the  finely  granular,  being  often 
dark  brown,  owing  to  presence  of  altered  blood-pigment. 
They  are  usually  shorter  and  more  irregular  in  outline, 
and  more  frequently  have  irregularly  broken  ends. 

(5)  Fatty  Casts. — Small  droplets  of  fat  may  at  times 
be  seen  in  any  variety  of  cast.  Those  in  which  the  drop- 
lets are  numerous  are  called  fatty  casts  (Figs.  62,  63  and 
81).     The  fat-globules  are  not  difficult  to  recognize. 


MICROSCOPIC   EXAMINATION  223 

Staining  with  osmic  acid  or  Sudan  III  (see  p.  210)  will 
remove  any  doubt  as  to  their  nature. 

The  granules  and  fat-droplets  seen  in  casts  are  prod- 
ucts of  epithelial  degeneration.  Granular  and  fatty 
casts,  therefore,  always  indicate  partial  or  complete  dis- 
integration of  the  renal  epithelium.  The  finely  granular 
variety  is  the  least  significant,  and  is  found  when  the 
epithelium  is  only  moderately  affected.  Coarsely 
granular,  and  especially  fatty  casts,  if  present  in  con- 
siderable numbers,  point  toward  a  serious  parenchyma- 
tous nephritis. 

(6)  Casts  Containing  Organized  Structures.^Cells 
and  other  structures  are  frequently  seen  adherent  to  a 
cast  or  embedded  within  it  (see  Figs.  60  and  61). 
When  numerous,  they  give  name  to  the  cast. 

(a)  Epithelial  casts  contain  epithelial  cells  from  the 
renal  tubules.  The  cells  vary  in  size  and  are  often 
flattened,  oval,  or  elongated.  They  may  be  recognized 
as  epithelial  cells  by  irrigating  with  dilute  acetic  acid, 
which  usually  brings  out  the  nucleus  clearly.  Epithelial 
casts  always  imply  desquamation  of  epithelium,  which 
rarely  occurs  except  in  parenchymatous  inflammations 
(see  Figs.  80  and  81) .  When  the  cells  are  well  preserved 
they  point  to  acute  nephritis. 

(b)  Blood-casts  contain  red  blood-corpuscles,  usually 
much  degenerated  (Figs.  64,  65,  and  80).  They  always 
indicate  hemorrhage  into  the  tubules,  which  is  most 
common  in  acute  nephritis  or  an  acute  exacerbation  of 
a  chronic  nephritis. 

(c)  Pus-casts  (see  Fig.  82),  composed  almost  wholly 
of  pus-corpuscles,  are  uncommon,  and  point  to  a  chronic 
suppurative  process  in  the  kidney.     Casts  containing 


224 


THE    URINE 


a  few  pus-corpuseles,  either  alone  or  in  combination 
with  epithelial  or  red  blood  cells  are  common.  In  these 
the  pus-cells  have  no  special  significance. 


Pig.  64. — Two  blood-casts,  one  containing  a  leukocyte;  six  free 
red  blood -cells;  and  two  renal  epithelial  cells.  From  the  urine  of  a 
child  with  acute  nephritis  (  X  300). 


Fig.  65. — Red  blood-corpuscles  and  blood-casts  (courtesy  of  Dr.  A. 
Scott)  (obj.  4  mm.)  (Boston). 

(d)  True  bacterial  casls  are  rare.  They  indicate  a 
septic  condition  in  the  kidney.  Bacteria  may  permeate 
a  cast  after  the  urine  is  voided. 


MICRdSCOPIC    EXAMINATION  22$ 

Structures  Likely  to  be  Mistaken  for  Casts. — (i) 
Mucous  Threads. — Mucus  frequently  appears  in  the 
form  of  long  strands  which  slightly  resemble  hyaline 
casts  (Fig.  66).  They  are,  however,  more  ribbon-like, 
have  less  well-defined  edges,  and  usually  show  faint 
longitudinal  striations.  Their  ends  taper  to  a  point  or 
are  split  or  curled  upon  themselves,  and  are  never  evenly 
rounded,  as  is  commonly  the  case  with  hyaline  casts. 


*)^ 

1 

i"^"**^-*^ 

-     i 

/^^ 

/ 

\\  V 

^^'*'r>>.... 

rJF 

'^^ 

^m-. 

'    \j^^-:. 

■>^, 

Fig.  60.  —  H>  aiiiie  and  granular  casts,  mucous  threads,  and  cylindroids. 
There  are  also  a  few  epithelial  cells  from  the  bladder  (Wood). 


Such  threads  form  a  part  of  the  nubecula  of  normal 
urine,  and  are  especially  abundant  when  calcium  oxalate 
crystals  are  present.  When  there  is  an  excess  of  mucus, 
as  in  irritations  of  the  urinary  tract,  every  field  may  be 
filled  with  an  interlacing  meshwork. 

Mucous  threads  are  microscopic  and  should  not  be 
confused  with  urethral  shreds  or  ''gonorrheal  threads," 
which  are  macroscopic,  0.5  to  i  cm.  long,  and  consist  of 

15 


226  THE   URINE      ' 

a  matrix  of  mucus  in  which  many  epithelial  and  pus-cells 
are  embedded. 

(2)  Cylindroids.- — This  name  is  sometimes  given  to 
the  mucous  threads  just  described,  but  is  more  properly 
apphed  to  certain  peculiar  structures  more  nearly  allied 
to  casts.  They  resemble  hyaline  casts  in  structure,  but 
differ  in  being  broader  at  one  end  and  tapering  to  a 
slender  tail,  which  is  often  twisted  or  curled  upon  itself 
(Fig.  66).  They  frequently  occur  in  the  urine  along 
with  hyaline  casts,  especially  in  irritations  of  the  kidney, 
and  have  practically  the  same  significance. 

(3)  Masses  of  amorphous  urates,  or  phosphates,  or 
very  small  crystals  (Fig.  67),  which  accidentally  take  a 


1 

,c 

% 

i 

w 

^^ 

.j 

L„ 

0 

^.lA<'/pe./7/7e//^ 

Fig.  67. — Two  pseudo-casts,  one  composed  of  calcium  oxalate  crystals, 
one  of  uric  acid  (  X  300). 

cylindric  form,  or  shreds  of  mucus  covered  with  granules, 
closely  resemble  granular  casts.  Application  of  gentle 
heat  or  appropriate  chemicals  will  serve  to  differentiate 
them.  When  urine  contains  both  mucus  and  granules, 
large  numbers  of  these  "pseudocasts,"  all  lying  in  the 
same  direction,  can  be  produced  by  slightly  moving  the 
cover-glass  from  side  to  side.     It  is  possible — as  in  urate 


MICROSCOPIC    EXAMINATION  227 

infarcts  of  infants — for  urates  to  be  molded  into  cylin- 
dric  bodies  within  the  renal  tubules. 

(4)  Hairs  and  fibers  of  wool,  cotton,  etc.  These 
could  be  mistaken  for  casts  only  by  beginners.  One 
can  easily  become  familiar  with  their  appearance  by 
suspending  them  in  water  and  examining  with  the  micro- 
scope (see  Fig.  78). 

(5)  Hyphae  of  molds  are  not  infrequently  mistaken 
for  hyaline  casts.  Their  higher  degree  of  refraction, 
their  jointed  or  branching  structure,  and  the  accom- 
panying spores  will  differentiate  them  (see  Fig.  79). 

2.  Epithelial  Cells. — A  few  cells  from  various  parts 
of  the  urinary  tract  occur  in  every  urine.  A  marked 
increase  indicates  some  pathologic  condition  at  the  site 
of  their  origin.  It  is  sometimes,  but  by  no  means  always, 
possible  to  locate  their  source  from  their  form.  One 
should,  however,  be  extremely  cautious  about  making 
any  definite  statement  as  to  the  origin  of  any  individual 
cell.  Most  cells  are  much  altered  from  their  original 
shape.  Any  epithelial  cell  may  be  so  granular  from 
degenerative  changes  that  the  nucleus  is  obscured. 
Most  of  them  contain  fat-globules.  They  are  usually 
divided  into  three  groups : 

(i)  Small,  round  or  polyhedral  cells  are  about  the 
size  of  pus-corpuscles,  or  a  little  larger,  with  a  single 
round  nucleus.  Such  cells  may  come  from  the  deeper 
layers  of  any  part  of  the  urinary  tract.  They  are  uncom- 
mon in  normal  urine.  When  they  are  polygonal  in 
shape,  rather  dark  in  color,  very  granular,  and  contain 
a  comparatively  large  nucleus  (Fig.  68) ,  they  probably 
come  from  the  renal  tubules,  but  their  origin  in  the 
kidney  is  not  proved  unless  they  are  found  embedded 


228  THE    URINE 

in  casts.  In  chronic  passive  congestion  of  the  kidney 
and  in  renal  infarction  some  of  these  cells  may  contain 
yellow  granules  of  altered  blood-pigment.  They  are 
analogous  to  the  "heart-failure  cells"  of  the  sputum 
(see  p.  70),  Renal  cells  are  abundant  in  parenchyma- 
tous nephritis,  especially  the  acute  form.  They  are 
nearly   always   fatty— -most   markedly   so  in  chronic 


Fig.  68. — Renal  epithelial  cells  from  nephritic  urine.     The  four  cells 
below  show  different  grades  of  fatty  degeneration  (X  475). 

parenchymatous  nephritis,  where  their  substance  is 
sometimes  wholly  replaced  by  fat-droplets  ("compound 
granule  cells")  (see  Figs.  63,  68,  80  and  81). 

(2)  Irregular  cells  are  considerably  larger  than  the 
preceding.  They  are  round,  pear  shaped,  or  spindle 
shaped,  or  may  have  tail-like  processes,  and  are  hence 
named  large  round,  pyriform,  spindle,  or  caudate  cells 
respectively.  Each  contains  a  round  or  oval,  distinct 
nucleus.  Their  usual  source  is  the  deeper  layers  of  the 
urinary  tract,  especially  of  the  bladder.  Caudate 
,  forms  apparently  come  most  commonly  from  the  pelvis 
of  the  kidney  (see  Figs.  69,  70,  h  and  82). 


MICROSCOPIC    EXAMINATION  229 

(3)  Squamous  or  pavement  cells  are  large  flat  cells, 
each  with  a  small,  distinct  round  or  oval  nucleus  (Fig. 
70,  a).     They  are  derived  from  the  superficial  layers 


Fig.  69. — Caudate  epithelial  cells  from  pelvis  of  kidney  (Jakob). 


Fig.  70. — Epithelial  cells  from  urethra  and  bladder:  a.  Squamous  cells 
from  superficial  layers;  b,  irregular  cells  from  deeper  layers  (Jakob). 

of  the  ureters,  bladder,  urethra,  or  vagina,  and  when 
desquamation  is  active,  appear  in  stratified  masses. 


230 


THE    URINE 


Squamous  cells  from  the  bladder  are  generally  rounded, 
while  those  from  the  vagina  are  larger,  thinner,  and 
more  angular.  Great  numbers  of  these  vaginal  cells, 
together  with  pus-corpuscles,  may  be  present  when 
leukorrhea  exists  (Fig,  71). 


Fig.  71. — -Squamous    epithelial  cells,  pus-corpviscles  and  bacteria  in 
urine;  vaginal  contamination  (  X  300). 


3.  Pus=corpuscles. — A  very  few  leukocytes  are 
present  in  normal  urine.  They  are  more  abundant  when 
mucus  is  present.  An  excess  of  leukocytes,  mainly  of 
the  polymorphonuclear  neutrophilic  variety,  with  albu- 
min, constitutes  pyuria — pus  in  the  urine.  At  times 
numerous  mononuclear  cells  (lymphocytes)   are  seen. 

When  at  all  abundant,  pus  forms  a  white  sediment 
resembling  amorphous  phosphates  macroscopically .  Un- 
der the  microscope  the  corpuscles  appear  as  very  granu- 
lar cells,  about  twice  the  diameter  of  a  red  blood-cor- 
puscle (Figs.  72  and  83).  The  granules  are  partly  the 
normal  neutrophilic  granules,  partly  granular  products 
of  degeneration.     In  freshly  voided  urine  many  exhibit 


MICROSCOPIC   EXAMINATION  23 1 

ameboid  motion,  assuming  irregular  outlines.  Each 
contains  one  irregular  nucleus  or  several  small,  rounded 
nuclei.  The  nuclei  are  obscured  or  entirely  hidden  by 
the  granules,  but  may  be  brought  clearly  into  view  by 
running  a  little  acetic  acid  under  the  cover-glass.  This 
enables  one  to  easily  distinguish  pus-corpuscles  from 


t 


® 
® 


®    ®    ® 
M     ^A     ^      ®      ®@ 

Fig.   72. — Pus-corpuscles:  a.  As   ordinarily  seen;  b,  ameboid  corpus- 
cles; c,  showing  the  action  of  acetic  acid  (Ogden). 

small  round  epithelial  cells,  which  resemble  them  in  size, 
but  have  a  single,  rather  large,  round  nucleus.  In  de- 
composing urine  pus  is  often  converted  into  a  gelatinous 
mass  which  gives  the  urine  a  ropy  consistence. 

Pyuria  indicates  suppuration  in  some  part  of  the 
urinary  tract — urethritis,  cystitis,  pyelitis,  etc. — or  may 
be  due  to  contamination  from  the  vagina,  in  which  case 
many  vaginal  epithelial  cells  will  also  be  present.  Of 
these  conditions  chronic  cystitis  usually  gives  by  far 
the  greatest  amount  of  pus.  In  general,  the  source  of 
the  pus  can  be  determined  only  by  the  accompanying 
structures  (epithelia,  casts)  or  by  the  clinical  signs.  A 
considerable  amount  of  pus,  appearing  suddenly,  usu- 
ally originates  from  a  ruptured  abscess. 

A  fairly  accurate  idea  of  the  quantity  of  pus  from  day 
to  day  may  be  had  by  shaking  the  urine  thoroughly  and 


232  THE    URINE 

counting  the  number  of  corpuscles  per  cubic  millimeter 
upon  the  blood-counting  slide.  A  drop  of  the  urine 
is  placed  directly  upon  the  slide.  Dilution  is  seldom 
necessary.  The  urine  must  not  be  alkaline  or  the  cor- 
puscles will  adhere  in  clumps. 

Pus  always  adds  a  certain  amount  of  albumin  to  the 
urine,  and  it  is  often  desirable  to  know  whether  the 
abumin  present  in  a  given  specimen  is  due  solely  to 
pus.  It  has  been  estimated  that  80,000  to  100,000  pus- 
corpuscles  per  cubic  millimeter  add  about  o.i  per  cent, 
of  albumin.  If  albumin  is  present  in  much  greater  pro- 
portion than  this,  the  excess  is  probably  derived  from  the 
kidney. 

4.  Red  BIood=corpuscles. — Urine  which  contains 
blood  is  always  albuminous.  Very  small  amounts  do  not 
alter  its  macroscopic  appearance.  Larger  amounts  alter 
it  considerably.  Blood  from  the  kidneys  is  generally 
intimately  mixed  with  the  urine  and  gives  it  a  hazy 
reddish  or  brown,  "smoky"  color.  When  from  the 
lower  urinary  tract,  it  is  not  so  intimately  mixed  and 
settles  more  quickly  to  the  bottom,  the  color  is  brighter, 
and  small  clots  are  often  present.  A  further  clue  to 
the  site  of  the  bleeding  may  sometimes  be  gained  by 
having  the  patient  void  his  urine  in  three  separate 
portions.  If  the  blood  be  chiefly  in  the  first  portion, 
the  bleeding  point  is  probably  in  the  urethra;  if  in  the 
last,  it  is  probably  in  the  bladder.  If  the  blood  is  uni- 
formly mixed  in  all  three  portions,  it  probably  comes  from 
the  kidney  or  ureter.  Microscopically  the  presence  of 
tube-casts  or  of  considerable  numbers  of  epithelial 
cells  of  the  renal  type  would  be  suggestive,  while  the 


MICROSCOPIC   EXAMINATION  233 

presence  of  blood-casts  would  of  course  point  definitely 
to  hemorrhage  into  the  kidney  tubules. 

Red  blood-corpuscles  are  not  usually  difficult  to 
recognize  with  the  microscope.  When  very  fresh,  they 
have  a  normal  appearance,  being  yellowish  disks  of  uni- 
form size.  When  they  have  been  in  the  urine  any  con- 
siderable time,  their  hemoglobin  may  be  dissolved  out, 
and  they  then  appear  as  faint  colorless  circles  or 
"shadow  cells,"  and  are  more  difficult  to  see  (Fig.  73; 


•% 

n 

O'-- 

)     ' 

A 

0 

I.'- 

Fig.  73. — Red  blood-corpuscles  in  urine:  yl ,  shadow  cells  from  a  case 
of  nephritis;  B,  fresh  red  corpuscles;  C,  crenated  corpuscles  in  a  urine  of 
high  specific  gravity  (  X  475)- 

see  also  Figs.  64,  65  and  80).  They  are  apt  to  be 
swollen  in  dilute  and  crenated  in  concentrated  urines. 
The  microscopic  findings  may  be  corroborated  by 
chemic  tests  for  hemoglobin,  although  the  microscope 
may  show  a  few  red  corpuscles  when  the  chemic  tests 
are  negative. 

When  not  due  to  contamination  from  menstrual  dis- 
charge, blood  in  the  urine,  or  hematuria,  is  always  patho- 
logic, although  not  always  of  serious  import.     A  few 


234  THE    URINE 

red  blood-corpuscles  may  be  found  after  strenuous 
exercise.  Blood  comes  from  the  kidney  tubules  in  severe 
hyperemia,  in  acute  nephritis  and  acute  exacerbations  of 
chronic  nephritis,  and  in  renal  tuberculosis  and  malig- 
nant disease.  Renal  hematuria  may  also  be  a  manifes- 
tation of  the  "hemorrhagic  diseases"  and  an  "idiopathic 
hematuria,"  probably  of  nervous  origin,  has  been  ob- 
served. The  urine  of  healthy  infants  frequently  contains 
red  blood-corpuscles  for  weeks  at  a  time.  This  has 
been  attributed  to  slight  toxic  injury  to  the  kidneys. 
Blood  comes  from  the  pelvis  of  the  kidney  in  renal  calculus 
(see  Fig.  82),  and  is  then  usually  intermittent,  small  in 
amount,  and  accompanied  by  a  little  pus  and  perhaps 
crystals  of  the  substance  forming  the  stone.  Consider- 
able hemorrhages  from  the  bladder  may  occur  in  vesical 
calculus,  tuberculosis,  and  new  growths.  Small  amounts 
of  blood  generally  accompany  acute  cystitis.  In  Africa 
the  presence  of  Schistosomum  hcematobium  in  the  veins 
of  the  bladder  is  a  common  cause  of  hemorrhage  (Egyp- 
tian hematuria). 

5.  Spermatozoa  are  generally  present  in  the  urine 
of  men  after  nocturnal  emissions,  after  epileptic  convul- 
sions, and  in  spermatorrhea.  They  may  be  found  in  the 
urine  of  both  sexes  following  coitus.  They  are  easily 
recognized  from  their  characteristic  structure  (Fig.  74). 
The  4-mm.  objective  should  be  used,  with  subdued  light 
and  careful  focusing. 

6.  Bacteria. — Normal  urine  is  free  from  bacteria  in 
the  bladder,  but  becomes  contaminated  in  passing 
through  the  urethra.  Various  non-pathogenic  bacteria 
are  always  present  in  decomposing  urine.  In  suppura- 
tions of  the  urinary  tract  pus-producing  organisms  may 


MICROSCOPIC   EXAMINATION  235 

be  found.  In  many  infectious  diseases  the  specific 
bacteria  may  be  eliminated  in  the  urine  without  pro- 
ducing any  local  lesion.  Typhoid  bacilli  have  been 
known  to  persist  for  months  and  even  years  after  the 
attack. 

Bacteria  produce  a  cloudiness  which  will  not  clear 
upon  filtration.     They  are  easily  seen  with  the  4-mm. 


e 

( 

9 

c 

r 

0 

0 

--C 

® 

c 

^ 

C 

0 

4 

Oy 

■^ 

V 

0    , 

r 

0 

p 

0 

^ 

1 

Fig.   74. — Spermatozoa  in  urine  (Ogden). 

objective  in  the  routine  microscopic  examination. 
Ordinarily,  no  attempt  is  made  to  identify  any  but  the 
tubercle  bacillus  and  the  gonococcus.  Others  must  be 
studied  by  cultural  methods,  the  urine  being  carefully 
obtained  by  catheter  and  received  directly  into  a  sterile 
bottle  or  test-tube 

Tubercle  bacilli  are  nearly  always  present  in  the  urine 
when  tuberculosis  exists  in  any  part  of  the  urinary  tract 
and  are  often  present  in  general  miliary  tuberculosis, 


236  THE   URINE 

but  may  be  difficult  to  find,  especially  when  the  urine 
contains  little  or  no  pus. 

Detection  of  Tubercle  Bacilli  in  Urine. — In  order  to  avoid 
the  smegma  bacillus  the  urine  should  be  obtained  aseptically 
by  catheter  after  careful  cleansing  of  the  parts,  or  by  having 
the  patient  void  urine  in  three  portions,  only  the  last  being 
used  for  the  examination. 

1.  Centrifugalize  thoroughly,  or,  better,  treat  by  the 
following  method,  recommended  by  Brown: 

(a)  Acidify  100  c.c.  of  the  urine  with  30  per  cent,  acetic 
acid. 

(b)  Add  2  c.c.  of  5  per  cent,  tannic  acid  solution. 

(c)  Place  in  ice  chest  for  twenty-four  hours. 

(d)  Centrifugalize,  pipet  off  the  supernatant  fluid  and 
re-dissolve  the  sediment  with  dilute  acetic  acid. 

(e)  Centrifugalize  thoroughly  once  more. 

2.  Make  thin  smears  of  the  sediment,  adding  a  little 
egg-albumen  if  necessary  to  make  the  smear  adhere  to  the 
glass;  dry,  preferably  in  the  incubator  for  three  hours, 
and  fix  in  the  usual  way. 

3.  Stain  thoroughly  with  carbol-fuchsin  in  the  usual 
way  (see  p.  77). 

4.  Wash  in  water,  and  then  in  5  to  20  per  cent,  nitric 
acid,  until  only  a  faint  pink  color  remains. 

5.  Wash  in  water. 

6.  Soak  in  alcohol  fifteen  minutes  or  longer.  This  de- 
colorizes the  smegma  bacillus  (see  p.  82),  which  is  often 
present  in  the  urine,  and  might  easily  be  mistaken  for  the 
tubercle  bacillus.  Some  strains  of  the  smegma  bacillus 
are  very  resistant  to  alcohol.  It  is  therefore  best  to  avoid 
it  altogether  by  examining  only  catheterized  specimens,  in 
which  case  this  step  may  be  omitted. 

7.  Wash  in  water. 


PLATE  IV. 


Tubercle  bacilli  in  urinary  sediment;   X  800  (Ogden). 


MICROSCOPIC    EXAMINATION  237 

8.  Apply  LofHer's  methylene  blue  solution  for  one-half 
minute. 

9.  Rinse  in  water,  dry  between  filter-papers,  and  examine 
with  the  oil-immersion  objective. 

A  careful  search  of  several  smears  may  be  necessary  to 
find  the  bacilli.  They  usually  lie  in  clusters  (see  Plate 
IV).  Failure  to  find  them  in  suspicious  cases  should  be 
followed  by  inoculation  of  guinea-pigs;  this  is  the  court  of 
last  appeal,  and  must  also  be  sometimes  resorted  to  in  order 
to  exclude  the  smegma  bacillus. 

In  gonorrhea,  gonococci  are  sometimes  found  within 
pus-cells  in  the  sediment,  but  more  commonly  in  the 
"gonorrheal  threads"  or  "floaters."  In  themselves, 
these  threads  are  by  no  means  diagnostic  of  gonorrhea. 
They  are  most  common  in  the  morning  or  after  mas- 
sage of  the  prostate.  Detection  of  the  gonococcus  is 
described  later  (see  p.  518).  Its  recognition  in  isolated 
pus-cells  in  the  urine  is  difficult  since  these  are  usually 
much  shrunken.  The  smears  should  be  thin  and 
quickly  dried. 

7.  Animal  parasites  are  rare  in  the  urine.  Hook- 
lets  and  scolicesof  T cenia  echinococcus  (Fig.  75)  and  larvae 
of  filarial  have  been  met.  In  Africa  the  ova,  and  even 
adults,  of  Schistosomum  hcBmatohhim  are  common, 
accompanying  "Egyptian  hematuria."  Trichomonas 
intesiinalis  is  a  not  uncommon  contamination.  This 
and  other  protozoa  may  be  mistaken  for  spermatozoa 
by  the  inexperienced. 

A  worm  which  is  especially  interesting  is  Anguillula 
aceti,  the  "vinegar  eel."  This  is  generally  present  in 
the  sediment  of  table  vinegar,  and  may  reach  the  urine 
through  use  of  vinegar  in  vaginal  douches,  or  through 


238 


THE   URINE 


contamination  of  the  bottle  in  which  the  urine  is  con- 
tained.    It  has  been  mistaken  for  Strongyloides  intes- 


FlG.  7$. — I,  Scolex  of  Taenia  echinococcus,  showing  crown  of  booklets; 
2,  scolex  and  detached  booklets  (obj.  4  mm.)  (Boston). 

tinalis  and  for  the  larval  filaria.     It  somewhat  resembles 
the   former   in    both   adult  and  embryo  stages.     The 


Fig.  76. — Embryo  of  "vinegar  eel"  in  urine,  from  contamination; 
length,  340  ix\  width,  15  yu.  An  epithelial  cell  from  bladder  and  three 
leukocytes  are  also  shown. 

young  embryos  have  about  the  same  length  as  the 
larvae  oi  Filaria  bancrofti,  but  are  nearly  twice  as  broad, 


MICROSCOPIC    EXAMINATION  239 

and  the  intestinal  canal  is  comparatively  easily  seen 
(compare  Figs.  76  and  188). 

For  fuller  descriptions  of  these  parasites  the  reader 
is  referred  to  Chapter  VI. 

C.  Extraneous  Structures 

The  laboratory  worker  must  familiarize  himself  with 
the  microscopic  appearance  of  the  more  common  of  the 


^ 

O*-' 

© 

__/:^ 

/T3* 

0 

3 

-' 

% 

^^ 

■^' 

^ 

♦ 

f: 

^^ 

Fig.   77. — Yeasts   and   calcrum    oxalate   crystals  in  a  urine  which  had 
been  preserved  for  two  weeks  with  boric  acid  (  X  450). 

numerous  structures  which  may  be  present  from  acci- 
dental contamination  (Fig.  78). 

Yeast-cells  are  smooth,  colorless,  highly  refractive, 
spheric  or  ovoid  cells.  They  sometimes  reach  the  size  of 
a  leukocyte,  but  are  generally  smaller  (Fig.  77).  They 
are  often  mistaken  by  the  inexperienced  for  red  blood- 
corpuscles  and  more  rarely  for  fat-droplets,  or  the 
spheric  crystals  of  calcium  oxalate,  but  are  distinguished 
by  the  facts  that  they  are  usually  ovoid  and  not  of  uni- 


240 


THE   URINE 


form  size;  that  they  tend  to  adhere  in  short  chains;  that 
small  buds  may  often  be  seen  adhering  to  the  larger  cells ; 
and  that  they  do  not  give  the  hemoglobin  test,  are  not 
stained  by  osmic  acid  or  Sudan  III,  but  are  colored 
brown  by  Lugol's  solution,  and  are  insoluble  in  acids 


Vw^ 


Fig.  78. — Extraneous  matters  found  in  urine:  a.  Flax-fibers;  6,  cot- 
ton-fibers; c,  feathers;  d,  hairs;  e,  potato-starch  granules;  /,  rice-starch 
granules;  g,  wheat-starch  granules;  h,  air-bubbles;  i,  muscular  tissue; 
k,  vegetable  tissue;  /,  oil-globules. 

and  alkalies.     Yeast-cells  multiply  rapidly  in  diabetic 
urine,  and  may  reach  the  bladder  and  multiply  there. 

Mold  fungi  (Fig.  79)  are  characterized  by  refractive, 
jointed,  or  branched  rods  (hyph^e),  often  arranged  in  a 
network,  and  by  highly  refractive  spheric  or  ovoid 
spores.     They  are  common  in  urine  which  has  stood 


MICROSCOPIC   EXAMINATION  24 1 

exposed  to  the  air.  Not  infrequently  a  spore  with  a 
short  hypha  growing  from  it  is  reported  as  a 
spermatozoon. 

Fibers  of  wool,  cotton,  linen,  or  silk,  often  colored, 
derived  from  towels,  the  clothing  of  the  patient,  or  the 
dust  in  the  air,  are  present  in  almost  every  urine.  Fat- 
droplets  are  most  frequently  derived  from  unclean 
bottles  or  oiled  catheters.    Starch-granules  may  reach 


Fig.  79.- — Aspergillus  from  urine  (Boston). 

the  urine  from  towels,  the  clothing,  or  dusting-powders. 
They  are  recognized  by  their  concentric  striations  and 
their  blue  color  with  iodin  solution.  Lycopodium 
granules  (see  Fig.  9)  may  also  reach  the  urine  from 
dusting-powders.  They  might  be  mistaken  for  the 
ova  of  parasites.  Bubbles  of  air  (see  Fig.  78,  h)  are 
often  confusing  to  beginners,  but  are  easily  recognized 
after  once  being  seen. 

Scratches  and  flaws  in  the  glass  of  slide  or  cover  are 
often  most  assiduously  studied  by  beginners,  and  are  not 
infrequently  reported  as  rare  crystals,  tube-casts,  or 
even  worms.     Dirt  upon  the  top  of  the  cover  (especially 

16 


242  THE    URINE 

when  this  is  taken  directly  from  the  original  box  without 
cleaning)  is  likewise  a  common  source  of  confusion. 
It  often  takes  the  form  of  crystals  which,  because  they 
are  more  prominent  than  the  structures  in  the  urine 
beneath  the  cover,  receive  the  student's  whole  atten- 
tion. Fibers  of  muscle  (Figs.  78,  i,  and  154)  and  other 
particles  which  are  evidently  of  fecal  origin  are  usually 
the  result  of  contamination,  but  may  rarely  be  present 
in  catheterized  specimens.  They  then  indicate  recto- 
vesical fistula. 

V.  THE  URINE  IN  DISEASE 

In  this  section  the  characteristics  of  the  urine  in  those 
diseases  which  produce  distinctive  urinary  changes  will 
be  briefly  reviewed. 

1.  Renal  Hyperemia. — Active  hyperemia  is  usually 
an  early  stage  of  acute  nephritis,  but  may  occur  inde- 
pendently as  a  result  of  temporary  irritation.  The  urine 
is  generally  decreased  in  quantity,  highly  colored,  and 
strongly  acid.  Albumin  is  always  present — usually  in 
traces  only,  but  sometimes  in  considerable  amount  for  a 
day  or  two.  The  sediment  contains  a  few  hyaline  and 
finely  granular  casts  and  an  occasional  red  blood-cell. 
In  very  severe  hyperemia  the  urine  approaches  that  of 
acute  nephritis. 

Passive  hyperemia  occurs  most  commonly  in  diseases 
of  the  heart  and  in  pregnancy.  The  quantity  of  urine 
is  somewhat  low  and  the  color  high,  except  in  preg- 
nancy. Albumin  is  present  in  small  amount  only.  As 
the  liver  is  usually  deranged  in  these  cases,  small  or 
moderate  amounts  of  urobilin  may  be  found.  The 
sediment  contains  a  very  few  hyaline  or  finely  granular 


THE   URINE   IN   DISEASE  243 

casts.     In  pregnancy  the  amount  of  albumin  should  be 
carefully  watched,  as  any  considerable  quantity,  and 


Fig.  80. — Sediment  from  acute  hemorrhagic  nephritis:  Red  blood- 
corpuscles;  leukocytes;  renal  cells  not  fattily  degenerated;  epithelial 
and  blood  casts  (Jakob). 


Fig.  81. — Sediment  from  chronic  parenchymatous  nephritis:  Hya- 
line (with  cells  attached),  waxy,  brown  granular,  fatty,  and  epithelial 
casts;  fattily  degenerated  renal  cells,  and  a  few  white  and  red  blood- 
corpuscles  (Jakob). 

especially  a  rapid  increase,  strongly  suggests  approach- 
ing eclampsia. 


244       '  THE    URINE 

2.  Nephritis. — The  various  degenerative  and  inflam- 
matory conditions  grouped  under  the  name  of  nephritis 
have  certain  features  in  common.  The  urine  in  all 
cases  contains  albumin  and  tube-casts,  and  in  all  well- 
marked  cases  shows  a  decrease  of  normal  solids,  espe- 
cially of  urea  and  the  chlorids.  In  chronic  nephritis, 
especially  of  the  interstitial  type,  there  may  be  remis- 
sions during  which  the  urine  is  practically  normal. 
The  degree  of  functional  derangement  is  probably  best 
ascertained  by  the  phenolsulphonephthalein  test  (see 
p.  112).  The  characteristics  of  the  different  forms  are 
well  shown  in  the  table  on  page  245. 

3.  Renal  Tuberculosis. — The  urine  is  pale,  usually 
cloudy.  The  quantity  may  not  be  affected,  but  is  apt 
to  be  increased.  In  early  cases  the  reaction  is  faintly 
acid  and  there  are  traces  of  albumin  and  a  few  renal  cells. 
In  advanced  cases  the  urine  is  alkaline,  has  an  offensive 
odor,  and  is  irritating  to  the  bladder.  Albumin  in  vary- 
ing amounts  is  always  present.  Pus  is  nearly  always 
present,  though  frequently  not  abundant.  It  is  gener- 
ally intimately  mixed  with  the  urine,  and  does  not 
settle  so  quickly  as  the  pus  of  cystitis.  Casts,  though 
present,  are  rarely  abundant,  and  are  obscured  by  the 
pus.  Small  amounts  of  blood  are  common.  Tubercle 
bacilli  are  nearly  always  present,  although  animal  in- 
oculation may  be  necessary  to  detect  them. 

4.  Renal  Calculus. — The  urine  is  usually  somewhat 
concentrated,  with  high  color  and  strongly  acid  reaction. 
Small  amounts  of  albumin  and  a  few  casts  may  be  pres- 
ent as  a  result  of  kidney  irritation.  Blood  is  frequently 
present,  especially  in  the  daytime  and  after  severe  ex- 
ercise.    Crystals  of  the  substance  composing  the  cal- 


THE    URINE   IN   DISEASE 


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THE   URINE   IN   DISEASE  247 

epithelial  cells  from  the  bladder — chiefly  large  round, 
pyriform,  and  rounded  squamous  cells.  Red  blood- 
corpuscles  are  often  numerous. 

In  chronic  cases  the  urine  is  generally  alkaline.  It  is 
pale  and  cloudy  from  the  presence  of  pus,  which  is  abun- 
dant and  settles  readily  into  a  viscid  sediment.  The 
sediment  usually  contains  abundant  amorphous  phos- 
phates and  crystals  of  triple  phosphate  and  ammonium 


Fig.  83. — Sediment  from  cystitis  (chronic):  Numerous  pus-cor- 
puscles, epithelial  cells  from  the  bladder,  and  bacteria;  a  few  red 
blood-corpuscles  and  triple  phosphate  and  ammonium  urate  crystals 
(Jakob). 

urate.     Vesical  epithelium,  is  common.     Numerous  bac- 
teria are  always  present  (Fig.  83). 
7.  Vesical  Calculus,  Tumors,  and  Tuberculosis. 

— These  conditions  produce  a  chronic  cystitis,  with  its 
characteristic  urine.  Blood,  however,  is  more  frequently 
present  and  more  abundant  than  in  ordinary  cystitis. 
With  neoplasms,  especially,  considerable  hemorrhages 
are  apt  to  occur.  Particles  of  the  tumor  are  sometimes 
passed  with  the  urine.     No  diagnosis  can  be  made  from 


248  THE    URINE 

the  presence  of  isolated  tumor  cells.     In  tuberculosis 
tubercle  bacilli  can  generally  be  detected. 

8.  Diabetes  Insipidus. — Characteristic  of  this  dis- 
ease is  the  continued  excretion  of  very  large  quantities 
of  pale,  watery  urine,  containing  neither  albumin  nor 
sugar.  The  specific  gravity  varies  between  i.coi  and 
1.005.  The  daily  output  of  solids,  especially  urea,  is 
increased. 

9.  Diabetes  Mellitus. — The  quantity  of  urine  is  very 
large.  The  color  is  generally  pale,  while  the  specific 
gravity  is  nearly  always  high — 1.030  to  1.050,  very 
rarely  below  1.020.  Sometimes  in  mild  or  early  cases 
the  urine  varies  little  from  the  normal  in  quantity,  color, 
and  specific  gravity.  The  persistent  presence  of  glu- 
cose is  the  essential  feature  of  the  disease.  The  amount 
of  glucose  may  be  small,  but  is  often  very  great,  some- 
times exceeding  8  per  cent.,  while  the  total  elimination 
may  exceed  500  gm.  in  twenty-four  hours.     It  may  be 

.absent  temporarily.  Acetone,  indicating  acidosis,  is 
generally  present  in  advanced  cases.  Diacetic  and 
oxybutyric  acids  may  be  present,  and  usually  warrant 
an  unfavorable  prognosis.  Accompanying  the  acidosis 
there  is  a  corresponding  increase  in  amount  of  ammo- 
nia which  may  be  taken  as-  an  index  of  the  degree  of 
acidosis. 


CHAPTER  III 
THE  BLOOD 

Preliminary  Considerations. — The  blood  consists  of 
a  fluid  of  complicated  and  variable  composition,  the 
plasma,  in  which  are  suspended  great  numbers  of  micro- 
scopic structures:  viz.,  red  corpuscles,  white  corpuscles, 
blood-platelets,  and  blood-dust. 

Red  corpuscles,  or  erythrocytes,  appear  as  biconcave 
disks,  red  when  viewed  by  reflected  light  or  in  thick 
layer,  and  straw  colored  when  viewed  by  transmitted 
light  or  in  thin  layer.  They  give  the  blood  its  red  color. 
They  are  cells  which  have  been  highly  differentiated  for 
the  purpose  of  carrying  oxygen  from  the  lungs  to  the 
tissues.  This  is  accomplished  by  means  of  an  iron-bear- 
ing protein,  hemoglobin,  which  they  contain.  In  the 
lungs  hemoglobin  forms  a  loose  combination  with  oxy- 
gen, which  it  readily  gives  up  when  it  reaches  the  tissues. 
Normal  erythrocytes  do  not  contain  nuclei.  They  are 
formed  from  preexisting  nucleated  cells  in  the  bone- 
marrow. 

If  a  small  drop  of  blood  be  taken  upon  a  clean  slide 
and  covered  with  a  clean  cover-glass  as  in  diagnosis  of 
malaria  (see  p.  355)  the  red  corpuscles  in  the  thicker 
portions  of  the  preparation  will  often  show  a  striking 
tendency  to  lie  with  overlapping  edges,  like  piles  of 
coins  which  have  been  tilted  over.  Formerly  much 
attention  was  paid  to  this  "rouleaux  formation"  as  a 

249 


250  THE  BLOOD 

point  in  diagnosis  of  certain  diseases,  but  it  is  now 
little  regarded.  Also,  in  such  preparations  of  fresh 
blood,  many  of  the  red  corpuscles  are  seen  to  be  glob- 
ular in  shape  and  covered  with  knob-  or  spine-like 
processes  (see  Fig.  73).  This  is  called  "crenation'^  and 
has  little  or  no  clinical  significance.  It  is  favored  by 
concentration  of  the  fluid  due  to  evaporation  at  the 
edge  of  the  cover.  Crenated  corpuscles  are  often  seen 
in  concentrated  urine  and  other  body-fluids  and  should 
always  be  recognized. 

White  corpuscles,  or  leukocytes,  are  less  highly  difl'er- 
entiated  cells.  There  are  several  varieties.  They  all 
contain  nuclei,  and  most  of  them  contain  granules  which 
vary  in  size  and  staining  properties.  They  are  formed 
chiefly  in  the  bone-marrow  and  lymphoid  tissues. 
Their  function  is  not  fully  understood.  It  appears  to 
be  concerned  chiefly  with  the  protection  of  the  body 
against  harmful  agencies,  in  part  through  phagocytosis, 
in  part  through  production  of  antitoxic  substances  and 
of  ferments  which  play  an  important  role  in  pathology. 

Blood-platelets,  or  blood-plaques,  are  colorless  or 
slightly  bluish,  sjpheric  or  ovoid  bodies,  usually  about 
one-third  or  one-half  the  diameter  of  an  erythrocyte, 
sometimes  even  as  large  as  an  erythrocyte.  They 
appear  to  be  constricted-off  portions  of  the  pseudopodia 
of  certain  giant  cells  of  the  bone  marrow.  Their  func- 
tion is  not  fully  known,  but  is- in  some  way  connected 
with  coagulation. 

The  blood-dust  of  Miiller  {"hemoconia'')  consists  of 
fine  granules  which  have  vibratory  motion.  The  larger 
granules  resemble  micrococci.  Little  is  known  of  them 
and  they  are  given  no  consideration  in  clinical  blood- 


SERUM  251 

examinations.  It  has  been  suggested  that  they  are 
granules  from  disintegrated  leukocytes. 

The  total  amount  of  blood  as  shown  by  the  new- 
method  of  Keith,  Rowntree  and  Geraghty,  averages 
about  one-twelfth  of  the  body-weight.  Little  attention 
is  paid  to  this  subject  in  clinical  work,  but  it  is  clear 
that  fluctuations  in  volume,  which  are  common  in  path- 
ologic conditions,  must  have  a  marked  influence  upon 
the  percentage  of  hemoglobin  and  the  blood-cell  counts. 

The  reaction  is  alkaline  to  litmus. 

The  color  is  due  to  the  presence  of  hemoglobin  in  the 
red  corpuscles,  the  difference  between  the  bright  red  of 
arterial  blood  and  the  purplish  red  of  venous  blood  de- 
pending upon  the  relative  proportions  of  oxyhemoglobin 
and  reduced  hemoglobin.  The  depth  of  color  depends 
upon  the  amount  of  hemoglobin.  In  very  severe  ane- 
mias the  blood  may  be  so  pale  as  to  be  designated  as 
"watery."  The  formation  of  carbon-monoxid-hemo- 
globin  in  coal-gas-poisoning  gives  the  blood  a  bright 
cherry-red  color;  while  formation  of  methemoglobin  in 
poisoning  with  potassium  chlorate  and  certain  other 
substances  gives  a  chocolate  color. 

The  clear,  pale,  straw-colored  fluid  which  remains 
after  coagulation  (see  p.  257)  and  separation  of  the  clot 
is  called  serum.  In  the  serum  are  found  the  numerous 
substances  which  the  tissues  elaborate  for  protection 
against  bacterial  and  other  harmful  agents.  In  most 
cases  these  substances,  or  "antibodies,"  are  elaborated 
only  when  the  harmful  agent  is  present  in  the  body, 
and  they  are  "specific,"  that  is,  they  are  effective  only 
against  the  one  disease  which  has  called  them  forth.  A 
test  for  the  presence  of  the  antibody  is,  therefore,  a 


252  THE   BLOOD 

test  for  the  existence  of  the  particular  disease.  The 
various  tests  based  upon  these  principles  have  within 
recent  years  become  a  very  important  part  of  clinical 
laboratory  work.  They  are  discussed  in  the  chapter 
upon  Serodiagnostic  Methods. 

Clinical  study  of  the  blood  may  be  discussed  under 
the  following  heads:  I.  Methods  of  obtaining  blood 
for  examination.  II.  Coagulation.  III.  Hemoglobin. 
IV,  Enumeration  of  erythrocytes.  V.  Color  index. 
VI.  Volume  index.  VII.  Enumeration  of  leukocytes. 
VIII.  Enumeration  of  platelets.  IX.  Study  of  stained 
blood.  X.  Blood  parasites.  XI.  Tests  for  recognition 
of  blood.  XII.  Less  frequently  used  methods.  XIII. 
Special  blood  pathology. 


Fig.  84. — Daland's  blood-lancet. 
I.  METHODS  OF  OBTAINING  BLOOD 

For  most  clinical  examinations  only  one  drop  of  blood 
is  required.  This  may  be  obtained  from  the  lobe  of  the 
ear,  the  palmar  surface  of  the  tip  of  the  finger,  or,  in  the 
case  of  infants,  the  plantar  surface  of  the  great  toe.  In 
the  case  of  the  ear,  the  edge  of  the  lobe,  not  the  side, 
should  be  punctured.  I^;i  general,  the  finger  will  be 
found  most  convenient.  With  nervous  children  the  ear 
is  preferable,  as  it  is  less  sensitive  and  its  situation  pre- 
vents their  seeing  what  is  being  done.  An  edematous 
or  congested  part  should  be  avoided;  also  a  cold,  appar- 
ently bloodless  one.     The  site  should  be  well  rubbed 


METHODS    OF    OBTAINING   BLOOD  253 

with  alcohol  to  remove  dirt  and  epithelial  debris  and  to 
increase  the  amount  of  blood  in  the  part.  After  allowing 
sufficient  time  for  the  circulation  to  equalize,  the  skin  is 
punctured  with  a  blood-lancet  (of  which  there  are  sev- 
eral patterns  upon  the  market)  or  some  substitute,  as  a 
large  Hagedorn  needle,  aspirating  needle,  trocar,  a  spicule 
of  glass,  or  a  pen  with  one  of  its  nibs  broken  off.  The 
Hagedorn  needle  may  be  recommended  as  being  cheap, 
easily  obtained  and  fully  as  efficient  as  an  expensive 
lancet.  As  suggested  by  Bass  it  may  be  fixed  in  the 
cork  of  a  small  vial  of  alcohol  and  thus  kept  immersed 
in  the  fluid.  Nothing  is  more  unsatisfactory  than  an 
ordinary  round  sewing-needle.  The  lancet  should  be 
cleaned  with  alcohol  before  and  after  using,  but  need 
not  be  sterilized.  The  puncture  is  practically  painless 
if  properly  done  with  a  sharp  needle.  It  is  made  ivith  a 
firm,  quick  stab,  which,  however,  must  not  be  so  quick 
nor  made  from  so  great  a  distance  that  its  site  and  depth 
are  uncertain.  The  depth  may  be  guarded  with  the 
thumb-nail  if  the  lancet  is  not  provided  with  a  guard, 
but  this  should  not  be  necessary.  The  first  drop  of 
blood  which  appears  should  be  wiped  away,  and  the 
second  used  for  examination.  The  skin  at  the  site  of 
the  puncture  must  be  dry  else  the  blood  will  not  form 
a  rounded  drop  as  it  exudes.  The  blood  should  not 
be  pressed  out,  since  this  dilutes  it  with  serum  from  the 
tissues ;  but  moderate  pressure  some  distance  above  the 
puncture  is  allowable. 

For  serologic,  bacteriologic  and  chemic  examinations 
a  larger  amount  of  blood  is  required.  When  lo  to  20 
drops  will  suffice  they  can  be  obtained  from  a  deep  punc- 
ture of  the  lobe  of  the  ear.     For  this,  a  spring-lancet 


254  THE   BLOOD 

(Fig.  85)  is  best.  Larger  amounts  are  usually  drawn 
from  a  vein  as  described  below.  For  some  purposes, 
particularly  in  children  when  puncture  of  a  vein  is  not 
practicable,,  the  blood  can  be  obtained  by  means  of  a 
"wet  cup." 

Method  of  Obtaining  Blood  from  a  Vein. — Prepare 
the  skin  at  the  bend  of  the  elbow  as  for  a  minor  operation  or 
simply  rub  well  with  alcohol  or  paint  with  tincture  of 
iodin.  The  iodin  is  efficient  as  a  germicide  but  makes 
it  difficult  to  see  the  vein. 

Bind  a  rubber  or  muslin  bandage  tightly  around  the 
upper  arm.  The  cuff  of  the  blood-pressure  apparatus 
answers  admirably.     Instead  of   a  bandage  it  will  often 


^ 


Fig.  85. — Spring-lancet. 

be  sufficient  for  an  assistant  or  even  the  patient  to  grasp 
the  upper  arm  firmly. 

Have  the  patient  open  and  close  the  fist  a  few  times  and 
when  the  veins  are  sufficiently  distended  insert  a  sterile 
hypodermic  needle  attached  to  a  sterile  syringe  into  any 
vein  that  is  prominent.  The  needle  should  be  large — 
about  19  to  21  gauge.  It  should  go  through  the  skin  about 
3^^  inch  from  the  vein  with  the  bevel  at  its  tip  upper- 
most and  should  enter  the  vein  obliquely  from  the  side  in 
a  direction  opposite  to  the  blood-current.  If  the  needle  is 
pushed  through  the  skin  directly  over  the  vein,  the  vein  is 
likely  to  roll  to  one  side  thus  escaping  the  needle.  Unless 
too  small  a  needle  is  used,  blood  will  begin  to  rise  in  the 
syringe  as  soon  as  the  needle  has  entered  the  vein.  Suc- 
tion is  rarely  or  never  necessary. 

When  sufficient  blood  is  obtained  the  bandage  is  first 


METHODS    OF    OBTAINING   BLOOD  255 

removed  and  the  needle  is  then  withdrawn,  this  order  being 
followed  to  avoid  a  hematoma.  It  is  usually  easy  to  secure 
5  to  ID  c.c.  of  blood.  The  procedure  causes  the  patient  sur- 
prisingly little  inconvenience,  seldom  more  than  does  an 
ordinary  hypodermic  injection.  There  is  rarely  any  diffi- 
culty in  entering  a  vein  except  in  children  and  in  adults 
when  the  arm  is  fat  and  the  veins  are  small.  If  desired, 
one  of  the  veins  about  the  ankle  can  be  used. 

Instead  of  a  syringe  it  is  convenient  to  use  a  large  glass 
tube  which  has  been  drawn  oxxl  at  the  ends  and  one  end 
ground  to  fit  a  "slip  on"  needle.     A  rubber  tube  like  that 


Fig.  86.^ — Method  of  obtaining  blood  from  a  vein. 

used  on  hemacytometer  pipets  may  be  attached  to  the 
other  end,  thus  allowing  of  aspiration  if  the  blood  does 
not  enter  readily.  This  little  instrument  (Fig.  86)  can  be 
made  by  any  glass  blower  at  a  cost  of  less  than  fifty  cents, 
and  several  of  them  can  be  kept  on  hand  in  large  cotton- 
plugged  test-tubes  sterilized  ready  for  use.  Other  devices 
for  securing  blood  from  a  vein  are  shown  in  Figs.  87,  88 
and  89  which  indicate  their  construction  in  sufficient  de- 
tail. They  possess  the  advantage  that  the  blood  can  be 
drawn  directly  into  any  desired  reagent  or  culture  medium 
(see  p.  347). 


256 


THE   BLOOD 


9 


V 


/•— ■ 


d— 


d- 


A 


^f 


U 


"7 


g—- 


g— 


Fig.  87.  Fig.  89. 

Fig.  87. — Mcjunkin's  device  for  obtaining  blood  for  a  blood  culture: 
a,  large  test  tube;  ft,  rubber  tube;  c,  hypodermic  needle;  d,  cotton,  which 
is  wrapped  around  the  rubber  tube  before  it  is  inserted;  e,  small  test- 
tube  used  as  a  protecting  cap;  /,  cotton;  g,  oxalate  solution  or  culture 
medium. 

Fig.  88. — Keidel's  vacuum  tube  for  collecting  blood  from  a  vein, 
consisting  of  a  sealed  ampoule,  a  needle  with  rubber  connection  and  a 
glass  cap.  After  the  needle  has  entered  the  vein  the  stem  of  the  am- 
poule is  crushed  within  the  rubber  connection  and  blood  enters  because 
of  the  vacuum.  Similar  tubes  containing  sterile  culture  media  are 
upon  the  market. 

Fig.  8g. — Device  for  drawing  blood  from  a  vein  using  a  large  test- 
tube,  a  50  c.c.  centrifuge  tube,  or  a  small  flask. 


COAGULATION  257 

11.  COAGULATION 

Coagulation  consists  essentially  in  the  transformation 
of  fibrinogen,  one  of  the  proteins  of  the  blood-plasma, 
into  fibrin  by  means  of  a  ferment  called  thrombin.  The 
presence  of  calcium  salts  is  necessary.  The  resulting 
cpagulum  is  made  up  of  a  meshwork  of  fibrin  fibrils  with 
entangled  corpuscles  and  platelets.  The  clear,  straw- 
colored  fluid  which  is  left  after  separation  of  the  coagu- 
lum  is  called  hlood-serum.  Normally,  coagulation  takes 
place  in  two  to  eight  minutes  after  the  blood  leaves  the 
vessels.  The  usual  time  is  about  four  and  one-half 
minutes.  The  time  is  affected  by  the  temperature,  the 
size  of  the  drop,  cleanliness  of  the  instruments,  and 
other  factors.  Clotting  is  more  rapid  when  blood  is 
squeezed  from  a  puncture  than  when  it  flows  freely, 
owing  to  admixture  with  tissue  juice.  For  this  reason, 
some  prefer  to  take  the  blood  from  a  vein.  Pathologic- 
ally, it  is  delayed  in  hemophilia,  purpura,  scurvy,  and 
icterus.  Estimation  of  coagulation  time  is  very  im- 
portant as  a  preliminary  to  operation  when  there  is  any 
reason  to  expect  dangerous  capillary  oozing,  as  in  ton- 
silectomies  and  in  operations  upon  jaundiced  persons. 
In  treatment,  calcium  salts,  especially  the  lactate  and 
acetate,  are  used  to  hasten  coagulation;  citric  acid,  to 
retard  it. 

For  certain  purposes,  notably  in  bacteriologic  and 
opsonic  work,  it  is  desirable  to  prevent  coagulation  of 
blood  which  has  been  withdrawn.  This  may  be  ac- 
complished by  receiving  it  directly  into  a  solution  of  i 
per  cent,  sodium  citrate  (or  ammonium  oxalate) 
in  normal  salt  solution  or  into  a  tube  containing  a 
very   little   finely  powdered  potassium  oxalate.     This 

17 


258 


THE  BLOOD 


precipitates  the  calcium  salts  which  are  necessary  to 
coagulation. 

There  are  many  methods  of  ascertaining  the  coagu- 
lation time  and  results  by  the  different  methods  are  not 
comparable  because  their  end-points — the  point  in  the 
process  when  coagulation  is  assumed  to  have  taken 
place — are  not  uniform.  It  is  there- 
fore well  to  adopt  a  single  method  for 
one's  routine  work.  The  simplest 
method  is  to  receive  several  drops  of 
blood  (well  rounded  drops  4  to  5  mm. 
^  in  diameter)    on  a  clean  slide  and  to 

draw  a  needle  through  one  or  another 
of  them  at  one-minute  intervals.     When 
the  clot  is  dragged  along  by  the  needle, 
\  coagulation    has    taken    place.     Duke 

uses  a  glass  slide  to  which  two  glass 
disks  5  mm.  in  diameter  are  cemented. 
Well-rounded  drops  of  blood  are  re- 
ceived on  the  disks  and  the  slide  is  in- 
verted across  the  top  of  a  glass  or  beaker 

Fig.  90. — Show- 
ing difference  in 
shape  of  blood- 
drops    before    and 

after    coagulation  the  shapc  of  the  drop  when  the  slide  is 

(Duke's  method),     -i     u  •  .-      ^  •.•         /t—  \ 

held  in  a  vertical  position  (Fig.  90). 
For  more  accurate  work  the  method  of  Russell  and 
Brodie  as  modified  by  Boggs  is  now  generally  used. 

Boggs'  Method. — The  instrument  is  shown  in  Fig.  91. 
The  bottom  of  the  box  (A)  and  the  cone  (B)  are  of  glass. 

The  instrument  must  be  absolutely  clean.  Obtain  the 
blood  from  a  freely  flowing  puncture.  When  a  large  drop 
has  formed,  touch  the  small  end  of  the  cone  to  its  surface. 


containing  water  at  40°C.  and  covered 
with  a  towel.     Coagulation  is  judged  by 


^COAGULATION 


259 


Fig.  91. — Boggs'  coagulation  instrument:  A,  chamber  with  glass  bot- 
tom; B,  glass  cone;  C,  tube  through  which  air  is  blown. 


Fig.  92. — Diagram  showing  the  direction  taken  by  red  corpuscles  in 
Boggs'  method  for  coagulation  time:  Radial  movement  of  the  cor- 
puscles, D,  indicates  the  end-point  (after  Boggs). 


26o  THE   BLOOD    # 

Quickly  invert  the  cone  into  the  box.  Place  the  instru- 
ment on  the  microscope  and  blow  puffs  of  air  against  the 
drop  of  blood  at  intervals  by  means  of  a  rubber  bulb 
attached  to  C,  meanwhile  watching  the  motion  of  the 
corpuscles  with  a  low  power  of  the  microscope.  Coagula- 
tion has  occurred  when  the  corpuscles  move  en  masse  in  a 
radial  direction  and  spring  back  to  their  original  position 
(Fig.  92,  D).  The  time  is  counted  from  the  first  appearance 
of  the  blood  from  the  puncture  to  the  end-point. 

III.  HEMOGLOBIN 

Hemoglobin  is  an  iron-bearing  protein  which  nor- 
mally occurs  in  the  circulating  blood  in  two  forms: 
oxyhemoglobin  chiefly  in  arterial  blood;  and  reduced 
hemoglobin  (more  correctly  called  simply  hemoglobin) 
chiefly  in  venous  blood.  Through  the  action  of  acids, 
alkalies,  oxidizing  and  reducing  substances,  heat,  and 
other  agencies  it  is  readily  converted  into  a  series  of 
derivative  compounds  which  can  be  distinguished  by 
means  of  the  spectroscope. 

Most  of  these  derivative  compounds  are  formed  only  in 
blood  which  has  left  the  vessels;  a  few,  however,  may  be 
produced  in  the  circulation. 

Methemoglobin  is  formed  in  the  circulating  blood  in  the 
rare  Gondition  known  as  "  enterogenous  cyanosis "  and  in 
pd^n^^  with  potassium  chlorate,  nitrites,  nitro-benzol, 
acetanilid,  phenacetin,  antipyrin,  and  other  substances. 
Clinically  there  is  marked  cyanosis,  and  in  severe  cases  the 
blood  has  a  chocolate-brown  color  when  withdrawn. 
Methemoglobinemia  is  easily  recognized  spectroscopically 
(seep.  370). 

Carbon  monoxid  hemoglobin,  formed  in  carbon  mon- 
oxid  poisoning,  gives  the  blood  a  brighter  red  color  than  is 


HEMOGLOBIN  26 1 

normal.  It  may,  in  some  cases,  be  identified  with  the 
spectroscope,  but  the  following  test  is  more  sensitive: 

Receive  about  ten  drops  of  blood  in  twenty  drops  of 
10  per  cent,  sodium  hydroxid  solution,  and  mix.  Blood 
containing  carbon  monoxid  remains  bright  red,  while 
normal  blood  takes  on  a  dirty  brownish-green  color. 

■  Normally  hemoglobin  is  confined  to  the  red  corpus- 
cles. When  it  is  dissolved  out  of  these  cells  and  appears 
in  the  plasma,  the  condition  is  known  as  hemoglohinemia. 
This  occurs  in  a  great  variety  of  conditions,  among 
which  may  be  mentioned:  severe  types  of  infectious 
diseases;  the  hemorrhagic  diseases  (scurvy,  etc.);  par- 
oxysmal hemoglobinuria;  severe  burns  and  frost  bites; 
and  poisoning  with  potassium  chlorate,  mushrooms,  etc. 

To  recognize  hemoglohinemia,  receive  a  little  blood 
in  a  small  test-tube  and  allow  it  to  stand  in  a  cool  place 
for  twenty-four  hours.  The  serum,  which  separates  after 
coagulation,  will  be  colored  red  or  pink  instead  of  pale 
yellow  as  is  normally  the  case. 

The  normal  amount  of  hemoglobin  is  usually  given 
as  about  14  Gm.  per  100  c.c.  of  blood.  The  absolute 
amount  is,  however,  seldom  estimated  clinically:  it  is 
the  relation  which  the  amount  present  bears  to  an 
arbitrarily-fixed  normal  that  is  determined.  Thus  the 
expression,  ''50  per  cent,  hemoglobin,"  when  used  clin- 
ically, means  that  the  blood  contains  50  per  cent,  of 
the  normal.  Theoretically,  the  normal  would  be  100 
per  cent.,  but  with  the  methods  of  estimation  in  gen- 
eral use  the  blood  of  healthy  adults  ranges  from  80 
to  105  per  cent. ;  these  figures  may,  therefore,  be  taken 
as  representing  normal  limits.  There  are,  moreover, 
marked  fluctuations  with  age  and  sex  which  must  be 


262 


THE  BLOOD 


taken  into  account  in  any  careful  case-study.  These 
are  well  shown  in  Fig.  93,  which  is  based  upon  William- 
son's careful  spectrophotometric  study  of  the  blood  of 
919  healthy  persons  in  Chicago. 


Fig.  93. — Diagram  showing  average  hemoglobin  values  for  both 
sexes  at  different  ages.  The  corresponding  percentages  on  the  Sahli 
hemoglobinometer  are  also  shown. 


The  custom  of  recording  hemoglobin  in  terms  of  per- 
centage of  the  normal  is  grossly  inaccurate  and  leads  to 
much  confusion.  From  what  has  just  been  said  it  is  clear 
that  no  single  normal  standard  can  be  applied  to  all  ages 


HEMOGLOBIN  263 

and  both  sexes.  The  situation  is  complicated  by  the 
fact  that  no  two  hemoglobinometers  use  the  same  standard. 
A  blood,  for  example,  which  reads  100  per  cent,  on  the 
Dare  instrument  will  read  about  80  per  cent,  on  the  Sahli. 
A  record  therefore  means  little  unless  one  knows  what 
instrument  was  used  and  the  age  and  sex  of  the  patient. 
This  confusion  could  be  avoided  if  records  were  made  in 
terms  of  the  actual  percentage  of  hemoglobin,  i.e.,  in 
grams  per  100  c.c.  of  blood,  and  this  will  doubtless  soon 
become  customary.  The  reading  on  any  type  of  instrument 
can  readily  be  converted  into  absolute  percentage  if  one 
knows  what  amount  of  hemoglobin  was  adopted  by  the 
makers  as  normal.  This  calculation  will  be  given  with 
the  description  of  the  various  instruments. 

Increase  of  hemoglobin,  or  hyperckromemia,  is  un- 
common, and  is  probably  more  apparent  than  real.  It 
accompanies  an  increase  in  number  of  erythrocytes,  and 
may  be  noted  in  change  of  residence  from  a  lower  to  a 
higher  altitude;  in  poorly  compensated  heart  disease 
with  cyanosis;  in  concentration  of  the  blood  from  any 
cause,  as  the  severe  diarrhea  of  cholera;  and  in  "idio- 
pathic polycythemia." 

Decrease  of  hemoglobin,  or  oligochromemia,  is  very 
common  and  important.  It  is  the  distinctive  and  most 
striking  feature  of  the  anemias  (see  p.  380).  In 
secondary  anemia  the  hemoglobin  loss  may  be  slight  or 
very  great.  In  mild  cases  a  slight  decrease  of  hemo- 
globin is  the  only  blood  change  noted.  In  very  severe 
cases,  especially  in  repeated  hemorrhages,  malignant 
disease,  and  infection  by  the  hookworm  and  Di- 
bothriocephalus  latus,  hemoglobin  may  fall  to  15  per 
cent.  Hemoglobin  is  always  diminished,  and  usually 
very  greatly,  in  chlorosis  (average  about  40  to  45  per 


264 


THE   BLOOD 


cent.),  pernicious  anemia  (average  about  20  to  25  per 
cent.),  and  leukemia  (usually  about  40  to  50  per  cent.). 
Estimation  of  hemoglobin  is  less  tedious  and  usually 
more  helpful  than  a  red  corpuscle  count.  It  offers 
the  simplest  and  most  certain  means  of  detecting  the 
existence  and  degree  of  anemia,  and  of  judging  the 


Pig.  94. — Von  Fleischl's  hemoglobinometer:  a.  Stand;  h,  narrow- 
wedge-shaped  piece  of  colored  glass  fitted  into  a  frame  (c),  which  passes 
under  the  chamber;  d,  hollow  metal  cylinder,  divided  into  two  com- 
partments, which  holds  the  blood  and  water;  e,  plaster-of-Paris  plate 
from  which  the  light  is  reflected  through  the  chamber;/,  screw  by  which 
the  frame  containing  the  graduated  colored  glass  is  rhoved;  g,  capillary 
tube  to  collect  the  blood;  h,  pipet  for  adding  the  water;  i,  opening 
through  which  may  be  seen  the  scale  indicating  percentage  of  hemo- 
globin. 


effect    of    treatment    in    anemic    conditions.     Pallor, 
observed  clinically,  does  not  always  denote  anemia. 

There  are  many  methods,  but  none  is  entirely  satis- 
factory. Those  which  are  most  widely  used  are  here 
described: 


HEMOGLOBIN  265 

I.  Von  Fleischl  Method. — The  apparatus  consists  of  a 
stand  somewhat  Hke  the  base  and  stage  of  a  microscope 
(Fig.  94).  Under  the  stage  is  a  movable  bar  of  colored  glass, 
shading  from  pale  pink  at  one  end  to  deep  red  at  the  other. 
The  frame  in  which  this  bar  is  held  is  marked  with  a  scale  of 
hemoglobin  percentages  corresponding  to  the  different 
shades  of  red.  By  means  of  a  rack  and  pinion  the  color- 
bar  can  be  moved  from  end  to  end  beneath  a  round  opening 
in  the  center  of  the  stage.  A  small  metal  cylinder,  which 
has  a  glass  bottom  and  which  is  divided  vertically  into  two 
equal  compartments,  can  be  placed  over  the  opening  in 
the  stage  so  that  one  of  its  compartments  lies  directly  over 
the  color-bar.  Accompanying  the  instrument  are  a 
number  of  short  capillary  tubes  in  metal  handles. 

Having  punctured  the  finger-tip  or  lobe  of  the  ear,  as  al- 
ready described,  wipe  ofif  the  first  drop  of  blood,  and  from  the 
second  fill  one  of  the  capillary  tubes.  Hold  the  tube  hori- 
zontally, and  touch  its  tip  to  the  drop  of  blood,  which  will 
readily  flow  into  it  if  it  be  clean  and  dry.  Avoid  getting  any 
blood  upon  its  outer  surface.  With  a  medicine-dropper 
rinse  the  blood  from  the  tube  into  one  of  the  compartments 
of  the  cylinder,  using  distilled  water,  and  mix  well.  Fill 
both  compartments  level  full  with  distilled  water,  and  place 
the  cylinder  over  the  opening  in  the  stage,  so  that  the  com- 
partment which  contains  only  water  lies  directly  over  the 
bar  of  colored  glass.  If  there  are  any  clots  in  the  hemo- 
globin compartment,  clean  the  instrument  and  begin  again. 

In  a  dark  room,  with  the  light  from  a  candle  reflected  up 
through  the  cylinder,  move  the  color-bar  along  with  a  jerk- 
ing motion  until  both  compartments  have  the  same  depth  of 
color.  The  number  upon  the  scale  corresponding  to  the 
portion  of  the  color-bar  which  is  now  under  the  cylinder 
gives  the  percentage  of  hemoglobin.  While  comparing 
the  two  colors,  place  the  instrument  so  that  they  will  fall 
upon  the  right  and  left  halves  of  the  retina,  rather  than 


266  THE  BLOOD 

upon  the  upper  and  lower  halves;  and  protect  the  eye  from 
the  light  with  a  cylinder  of  paper  or  pasteboard.  After 
use,  clean  the  metal  cylinder  with  water,  and  wash  the  capil- 
lary tube  with  water,  alcohol,  and  ether,  successively. 
Results  with  this  instrument  are  accurate  to  within  about 
5  per  cent. 

2.  The  Fleischl-Miescher  instrument,  a  modification  of 
the  preceding,  is  generally  considered  the  most  accurate 
hemoglobinometer  available.  It  is,  however,  better  adapted 
to  laboratory  use  than  to  the  needs  of  the  clinician.  De- 
tailed instructions  accompany  each  instrument.  The  chief 
differences  from  the  von  Fleischl  are:  (i)  The  blood  is  more 
accurately  measured  and  diluted,  a  pipet  like  that  ac- 
companying the  hemacytometer  being  used;  (2)  o.i  per 
cent,  solution  of  sodium  carbonate  is  used  instead  of  water 
for  diluting;  (3)  the  glass  bar  is  more  accurately  colored; 
(4)  there  are  two  cylindric  cells,  one  four-fifths  the  depth  of 
the  other;  and  (5)  the  cell  is  covered  with  a  glass  disk  and  a 
metal  cap  with  a  slit  through  which  the  reading  is  made. 
Each  Miescher  hemoglobinometer  is  accompanied  by  a 
chart  showing  the  actual  hemoglobin  values  in  grams  for 
each  reading  upon  that  particular  instrument. 

3.  The  Sahli  hemoglobinometer  (Fig.  95)  is  an  improved 
form  of  the  well-known  Gowers  instrument.  It  consists  of 
an  hermetically  sealed  comparison  tube  containing  a  suspen- 
sion of  acid  hematin,  a  graduated  test-tube  of  the  same 
diameter,  and  a  pipet  of  20-cu.  mm.  capacity.  The  two 
tubes  are  held  in  a  black  frame  with  a  white  ground-glass 
back. 

Place  decinormal  hydrochloric  acid  solution  in  the  gradu- 
ated tube  to  the  mark  lo.  Obtain  a  drop  of  blood  and  draw 
it  into  the  pipet  to  the  20cu.mm.  mark.  Wipe  off  the  tip 
of  the  pipet,  blow  its  contents  into  the  hydrochloric  acid 
solution  in  the  tube,  and  rinse  well.  The  hemoglobin  is 
changed  to  acid  hematin.     Place  the  two  tubes  in  the  com- 


HEMOGLOBIN  267 

partments  of  the  frame;  let  stand  one  minute;  and  dilute  the 
fluid  with  water  drop  by  drop,  mixing  after  each  addition, 
until  it  has  exactly  the  same  color  as  the  comparison  tube. 
The  graduation  corresponding  to  the  surface  of  the  fluid 
then  indicates  the  percentage  of  hemoglobin.  Mixing 
may  be  done  by  closing  the  tube  with  the  finger  and  invert- 
ing, but  care  should  be  exercised  to  see  that  none  of  the  fluid 
is  removed  by  adhering  to  the  finger.  Slightly  waxing  the 
finger  will  aid.  Decinormal  hydrochloric  acid  solution  is 
prepared  with  sufficient  accuracy  for  this  purpose  by  adding 


Fig.  95. — Sahli's  hemoglobinometer. 

12  c.c.  of  the  concentrated  acid  to  988  c.c.  distilled  water. 
A  little  chloroform  should  be  added  as  a  preservative. 

This  method  is  very  satisfactory  in  practice,  and  is  ac- 
curate to  within  5  per  cent.  Unfortunately,  not  all  the 
instruments  upon  the  market  are  well  standardized,  and  the 
comparison  tube  does  not  keep  its  color  unchanged  indefi- 
nitely. Usually,  however,  the  apparent  fading  is  due  to 
the  fact  that  the  hematin  is  in  suspension  and  settles  out 
when  the  instrument  lies  unused  for  some  time.  This  can 
be  remedied  by  inverting  the  tube  a  number  of  times.  Most 
tubes  contain  a  glass  bead  to  facilitate  mixing. 


268 


THE  BLOOD 


The  reading  upon  the  Sahh  multiplied  by  0.173  gives  the 
hemoglobin  value  in  grams  per  100  c.c.  of  blood.  The 
Kuttner  modification  of  this  instrument  (Fig.  31)  uses  a 
standard  color  tube  which  corresponds  to  15  Gm.  of  hemo- 
globin and  its  readings  should  be  multiplied  by  0.15  to 
find  the  actual  percentage. 

4.  Dare's  hemoglobinometer  differs  from  the  others  in 
using  undiluted  blood.  The  blood  is  allowed  to  flow  by 
capillarity  into  the  slit  between  two  small  plates  of  glass 
(Fig.  96,  W).     It  is  then  placed  in  the  instrument  and 


Fig.  96. — Dare's  hemoglobinometer. 

compared,  by  looking  through  the  tube,  U,  with  different 
portions  of  a  circular  disk  of  colored  glass  which  is  re- 
volved by  means  of  the  wheel,  R.  The  two  colors  appear 
side  by  side,  and  will  show  their  true  values  only  when 
viewed  in  a  darkened  room  by  the  light  of  the  candle,  Y. 
Usually  a  dark  corner  of  the  office  will  suffice.  The  read- 
ing is  taken  at  the  beveled  edge  of  a  slot  not  shown  in  the 
figure,  and  it  must  be  made  quickly,  before  clotting  takes 
place.  This  instrument  is  easy  to  use  and  to  clean,  and  is  one 
of  the  most  accurate.     One  hundred  per  cent,  on  the  scale  cor- 


HEMOGLOBIN 


269 


responds  to  13.77  Gm.  of  hemoglobin  per  100  c.c.  of 
blood.  When,  therefore,  it  is  desired  to  express  results 
in  actual  percentage,  the  readings  must  be  multiplied  by 

0-I377- 

5.  Tallqvist  Method. — The  popular  Tallqvist  hemo- 
globinometer  consists  simply  of  a  book  of  small  sheets  of  ab- 
sorbent paper  and  a  carefully  printed  scale  of  colors  (Fig.  97). 


Fig.  97. — Tallqvist's  hemoglobin  scale. 

Take  up  a  large  drop  of  blood  with  the  absorbent  paper, 
and  when  the  humid  gloss  is  leaving,  before  the  air  has  dark- 
ened the  hemoglobin,  compare  the  stain  with  the  color  scale. 
The  color  which  it  matches  gives  the  percentage  of  hemo- 
globin. Except  in  practised  hands,  this  method  is  accurate 
only  to  within  10  or  20  per  cent. 

Of  the  methods  given,  the  physician  should  select  the 
one  which  best  meets  his  needs.     With  any  method, 


270  THE  BLOOD 

practice  is  essertial  to  accuracy.  The  von  Fleischl  was 
for  many  years  the  standard  instrument,  but  is  now 
little  used.  For  accurate  work  the  best  instruments  are 
the  von  Fleischl-Miescher  and  the  Dare.  The  former 
is  essentially  a  laboratory  instrument.  The  Dare  is  easy 
to  use  and  to  clean,  and  is  probably  the  best  for  clinical 
work.  The  Sahli,  although  less  easy  to  use  and  proba- 
bly less  accurate,  is  inexpensive  and  is  very  satisfac- 
tory, provided  a  well-standardized  color-tube  is  obtained. 
The  Kuttner  modification  of  the  Sahli  is  an  improvement 
upon  the  original.  The  Tallqvist  scale  is  so  inexpen- 
sive and  so  convenient  that  it  should  be  used  by 
every  physician  at  the  bedside  and  in  hurried  office 
work;  but  it  should  not  supersede  the  more  accurate 
methods. 

IV.   ENUMERATION  OF  ERYTHROCYTES 

In  health  there  are  about  5,000,000  red  corpuscles  per 
cubic  millimeter  of  blood.  The  number  is  generally 
a  little  less — about  4,500,000 — in  women.  Age 
variations  in  general  follow  those  already  given  for 
hemoglobin.  Hawk  finds  the  normal  for  athletes  in 
training  to  be  5,500,000. 

Increase  of  red  corpuscles,  or  polycythemia,  is 
unimportant.  There  is  a  decided  increase  following 
change  of  residence  from  a  lower  to  a  higher  altitude, 
reaching  a  maximum  after  several  days  sojourn. 
The  increase,  however,  is  not  permanent.  In  a  few 
months  the  erythrocytes  return  to  nearly  their  original 
number.  At  the  University  of  Colorado  (altitude 
5400  feet)  the  average  for  healthy  medical  students 
is  about  5,600,000.     Three  views  have  been  offered  in 


ENUMERATION   OIT   EKYTHROCYTES  27 1 

explanation  of  this  effect  of  altitude:  (a)  Concentration 
of  the  blood,  owing  to  increased  evaporation  from  the 
skin;  (b)  altered  distribution  of  corpuscles,  the  reserve 
cells  in  the  splanchnic  vessels  being  thrown  into  the 
peripheral  circulation ;  (c)  new  formation  of  corpuscles, 
this  giving  a  compensatory  increase  of  aeration  surface. 
The  work  of  Schneider  at  Colorado  Springs  strongly 
supports  the  second  of  these  views,  although  the  third 
must  be  accepted  to  explain  the  moderate  permanent 
increase. 

Pathologically,  polycythemia  is  uncommon.  It  may 
occur  in:  (a)  Concentration  of  the  blood  from  severe 
watery  diarrhea;  (b)  chronic  heart  disease,  especially  the 
congenital  variety,  with  poor  compensation  and  cyano- 
sis; and  (c)  idiopathic  polycythemia,  which  is  considered 
to  be  an  independent  disease,  and  is  characterized 
by  dark  red  cast  of  countenance,  blood-counts  of 
7,000,000  to  10,000,000,  hemoglobin  120  to  150 per  cent., 
and  a  normal  number  of  leukocytes. 

Decrease  of  red  corpuscles,  or  oligocythemia.  Red 
corpuscles  and  hemoglobin  are  commonly  decreased 
together,  although  usually  not  to  the  same  extent. 

Oligocythemia  occurs  in  all  but  the  mildest  symp- 
tomatic anemias.  The  blood-count  varies  from  near 
the  normal  in  moderate  cases  down  to  1,500,000  in  very 
severe  cases.  There  is  always  a  decrease  of  red  cells  in 
chlorosis,  but  it  is  often  slight,  and  is  relatively  less  than 
the  decrease  of  hemoglobin.  Leukemia  gives  a  decided 
oligocythemia,  the  average  count  being  about  3,000,000. 
The  greatest  loss  of  red  cells  occurs  in  pernicious  anemia, 
where  counts  below  1,000,000  are  not  uncommon. 


272  THE  BLOOD 

Method  of  Counting. — The  most  widely  used  in- 
.  strument  for  counting  the  corpuscles  is  the  Thoma 
hemacytometer,  although  the  original  Thoma  ruling 
has  been  largely  displaced  by  more  convenient  ones. 
The  Burker  type  of  counting  chamber,  exemplified 
by  the  Burker  and  the  Levy  instruments,  is  a  decided 
improvement  and  when  supplied  with  the  Neubauer 
ruling  is  probably  the  most  satisfactory  of  all.  The 
Thoma-Metz  hemacytometer  is  convenient  for  routine 
working.  The  hematocrit,  which  at  one  time  promised 
much,  is  not  to  be  recommended  for  accuracy,  since  in 
anemia,  where  blood-counts  are  most  important,  the 
red  cells  vary  greatly  in  size  and  probably  also  in  elas- 
ticity. The  hematocrit  is,  however,  useful  in  deter- 
mining the  relative  volume  of  corpuscles  and  plasma 
(see  Volume  Index,  p.  285). 

Although  simple  in  principle,  accurate  counting  of 
blood-corpuscles  involves  a  technic  which  is  acquired 
only  after  considerable  practice.  Exact  and  fairly 
rapid  work  is  demanded.  Before  beginning,  one  should 
familiarize  himself  with  the  instrument  and  its  rul- 
ing, and  should  read  the  directions  carefully,  giving 
especial  attention  to  sources  of  error.  It  is  likewise 
an  advantage  to  practice  sucking  the  diluting  fluid 
into  the  pipet  and  stopping  it  at  a  predetermined  height 
and  also  to  practice  adjusting  the  cover-glass  after 
placing  a  drop  of  diluting  fluid  in  the  counting  chamber. 
In  our  class  work  we  have  found  Emerson's  plan  satis- 
factory: after  students  think  they  have  acquired  the 
technic  they  are  required  to  count  their  own  red  cor- 
puscles at  the  same  hour  upon  successive  days  until 
the  difference  between  successive  counts  falls  below 


ENUMERATION   OF    ERYTHROCYTES 


273 


200,000  cells.     Only  by  rigid  adherence  to  such  a  plan 
can  a  student  realize  his  inaccuracies. 

The  various  hemacytometers  are  here  described  in 
order,  but  detailed  directions  are  given  only  for  the 
Thoma  instrument.  Except  for  minor  variations  made 
necessary  by  differences  in  construction,  the  same  direc- 
tions apply  to  all  the  instruments. 

The  Thoma-Zeiss  instrument  consists  of  two  pipets  for 
diluting  the  blood  and  a  counting  chamber  (Fig.  98).     The 


0100mm 

f.. 

Fig.  98.- 


-Thoma-Zeiss  hemacytometer:  a.  Slide  used  in  counting;  b, 
sectional   view;   d,   red   pipet;   e,   white   pipet. 


rubber  tubes  which  come  with  the  pipets  are  too  short 
and  too  flexible  and  should  be  replaced.  For  this  purpose 
nothing  is  so  good  as  a  rubber  catheter.  The  counting  cham- 
ber is  a  glass  slide  with  a  square  platform  in  the  middle. 
In  the  center  of  the  platform  is  a  circular  opening,  in  which 
is  set  a  small  circular  disk  in  such  a  manner  that  it  is  sur- 
rounded by  a  "ditch,"  and  that  its  surface  is  exactly  o.i 
mm.   below   the  surface  of   the  square  platform.     Upon 

18 


2  74 


THE   BLOOD 


this  disk  is  ruled  a  square  millimeter,  subdivided  into  400 
small  squares.  Each  fifth  row  of  small  squares  has  double 
rulings  for  convenience  in  counting  (Fig.  99).  This  ruling, 
known  as  the  Thoma,  constitutes  the  central  square  milli- 
meter of  most  of  the  more  recent  forms,  such  as  the  Neu- 


)''l   Jc      0 


^^■ 


Fig.  99. — Essential  portion  of  Thoma  ruling  of  counting  chamber, 
showing  red  corpuscles  in  left  upper  corner.  This  ruling  constitutes 
the  central  portion  of  most  of  the  other  rulings. 


bauer  and  the  Turck  (see  Figs.  106,  107,  108).  A  thick 
cover-glass,  ground  perfectly  plane,  accompanies  the  count- 
ing chamber.  Ordinary  cover-glasses  are  of  uneven  sur- 
face, and  should  not  be  used  with  this  instrument.  For 
use  with  objectives  of  short  working  distance,  heavy  cover- 
glasses  can  be  obtained  with  a  flat-bottomed  excavation 


ENUMERATION    OF   ERYTHROCYTES 


275 


or  "well"  in  the  center.     This  combines  the  advantages 
of  a  thin  cover  with  the  rigidity  of  a  thick  one. 

It  is  evident  that,  when  the  cover-glass  is  in  place  upon 
the  platform,  there  is  a  space  exactly  o.i  mm.  thick  be- 
tween it  and  the  disk;  and  that,  therefore,  the  square 
millimeter  ruled  upon  the  disk  forms  the  base  of  a  space 
holding  exactly  o.i  cubic  millimeter. 

Technic. — To  count  the  red  corpuscles,  use  the  pipet  with 
loi  engraved  above  the  bulb.     It  must  be  clean  and  dry. 


Fig.   ioo. — Method  of  drawing  blood  into  the  pipet  (Boston).     The 
pipet  should  be  held  more  nearly  horizontal  than  here  represented. 


Puncture  the  skin,  wipe  off  the  first  drop  of  blood,  and  fill 
the  pipet  from  the  second,  sucking  the  blood  to  the  mark 
0.5  or  i.o,  according  to  the  dilution  desired.  While  doing 
this,  hold  the  pipet  horizontally  at  nearly  right  angles  to 
the  line  of  vision,  so  that  the  exact  height  of  the  column 
may  be  easily  seen.  The  side  of  the  tip  should  rest  against 
the  skin,  but  the  end  must  be  free.     Air-bubbles  will  enter 


276  THE   BLOOD 

if  the  drop  is  too  small  or  if  the  tip  is  not  kept  immersed. 
Should  the  blood  go  slightly  beyond  the  mark,  draw  it 
back  by  touching  the  tip  of  the  pipet  to  a  moistened  hard- 
kerchief.  Quickly  wipe  ofif  the  blood  adhering  to  the  tip, 
plunge  it  into  the  diluting  fluid,  and  suck  the  fluid  up  to  the 
mark  loi,  slightly  rotating  the  pipet  meanwhile.  At  this 
stage  it  is  best  to  hold  the  pipet  nearly  vertically  in  order 
to  avoid  inclusion  of  a  large  air  bubble  in  the  bulb.  This 
dilutes  the  blood  i :  200  or  i :  100,  according  to  the  amount  of 
blood  taken.  Except  in  cases  of  severe  anemia,  a  dilution 
of  1 :  200  is  preferable.  Close  the  ends  of  the  pipet  with  the 
fingers,  and  shake  vigorously  until  the  blood  and  diluting 
fluid  are  well  mixed,  keeping  the  pipet  horizontal  meanwhile. 
One  minute's  shaking  is  usually  sufficient.  Some  workers 
mix  by  rotating  the  pipet  rapidly  upon  its  long  axis. 

When  it  is  not  convenient  to  count  the  corpuscles  at  once, 
place  a  heavy  rubber  band  around  the  pipet  so  as  to  close 
the  ends,  inserting  a  small  piece  of  rubber-cloth  or  other 
tough,  non-absorbent  material,  if  necessary,  to  prevent  the 
tip  from  punching  through  the  rubber.  It  may  be  kept 
thus  for  twenty-four  hours  or  longer. 

When  ready  to  make  the  count,  clean  the  counting  cham- 
ber and  cover-glass,  and  place  a  sheet  of  paper  over  them  to 
keep  off  dust.  Mix  the  fluid  thoroughly  by  shaking;  blow  2 
or  3  drops  from  the  pipet,  wipe  off  its  tip,  and  then  place 
a  small  drop  (the  proper  size  can  be  learned  only  by  experi- 
ence) upon  the  disk  of  the  counting  chamber.  Adjust  the 
cover  immediately.  Hold  it  by  diagonal  corners  above  the 
drop  of  fluid  so  that  a  third  corner  touches  the  slide  and  rests 
upon  the  edge  of  the  platform.  Place  a  finger  upon  this  cor- 
ner, and,  by  raising  the  finger,  allow  the  cover  to  fall  quickly 
into  place.  If  the  cover  be  properly  adjusted,  faint  con- 
centric lines  of  the  prismatic  colors — Newton's  bands — 
can  be  seen  between  it  and  the  platform  when  the  slide  is 
viewed  obliquely.     They  indicate  that  the  two  surfaces  are 


ENUMERATION    OF    ERYTHROCYTES  277 

in  close  apposition.  If  they  do  not  appear  at  once,  slight 
pressure  upon  the  cover  may  bring  them  out.  Failure  to 
obtain  them  is  usually  due  to  dirty  slide  or  cover — both 
must  be  perfectly  clean  and  free  from  dust.  The  drop 
placed  upon  the  disk  must  be  of  such  size  that,  when  the 
cover  is  adjusted,  it  nearly  or  quite  covers  the  disk,  and 
that  none  of  it  runs  over  into  the  "ditch."  There  should 
be  no  bubbles  upon  the  ruled  area. 

The  following  is  an  easier  method  of  applying  the  cover: 
Place  a  drop  of  fluid  upon  the  ruled  disk.  The  size  of  the 
drop  is  of  no  great  consequence,  if  only  it  be  large  enough. 
Immediately  place  the  cover-glass  flat  upon  one  side  of  the 
platform  with  its  edge  close  to  the  drop  of  fluid,  and  hold  it 
firmly  down  with  the  two  index-fingers,  or  with  the  index- 
finger  and  middle  finger  of  the  right  hand.  Now  slide  it 
firmly  and  quickly  into  place.  If  the  drop  of  fluid  is  too 
large,  the  excess  will  be  caught  on  the  top  of  the  cover.  A 
moderately  thin  cover  is  best. 

Allow  the  corpuscles  to  settle  for  a  few  minutes,  and  then 
examine  with  a  low  power  to  see  that  they  are  evenly  dis- 
tributed. If  they  are  not  evenly  distributed  over  the  whole 
disk,  the  counting  chamber  must  be  cleaned  and  a  new  drop 
placed  in  it. 

Probably  the  most  satisfactory  objective  for  counting  is 
the  8-mm.  or  the  4-mm.  with  long  working  distance.  To 
understand  the  principle  of  counting,  it  is  necessary  to 
remember  that  the  square  millimeter  (400  small  squares) 
represents  a  capacity  of  o.i  c.c.  Find  the  number  of, 
"corpuscles  in  the  square  millimeter,  multiply  by  10  to  find 
the  number  in  i  cu.mm.  of  the  diluted  blood,  and  finally, 
by  the  dilution,  to  find  the  number  in  i  cu.mm.  of  undiluted 
blood.  Instead  of  actually  counting  all  the  corpuscles,  it  is 
customary  to  count  those  in  only  a  limited  number  of  small 
squares,  and  from  this  to  calculate  the  number  in  the  square 
millimeter.     Nearly  every  worker  has  his  own  method  of 


278 


THE  BLOOD 


doing  this.     The  essential  thing  is  to  adopt  a  method  and 
adhere  to  it. 

In  practice  a  convenient  procedure  is  as  follows:  With  a 
dilution  of  i :  200,  count  the  cells  in  80  small  squares,  and  to 
the  sum  add  4  ciphers;  with  dilution  of  i :  100,  count  40  small 
squares  and  add  4  ciphers.  Thus,  if  with  i :  200  dilution,  450 
corpuscles  were  counted  in  80  squares,  the  total  count  would 


Fig.   ioi. — Appearance  of  microscopic  field  in  counting  red  corpuscles. 
The  arrow  indicates  the  20  squares  to  be  counted. 


be  4,500,000  per  cu.mm.  This  method  is  sufficiently  accu- 
rate for  all  clinical  purposes,  provided  the  corpuscles  are 
evenly  distributed  and  2  drops  from  the  pipet  be  counted. 
It  is  convenient  to  count  a  block  of  20  small  squares,  as 
indicated  in  Fig.  loi,  in  each  corner  of  the  square  millimeter. 
Four  columns  of  5  squares  each  are  counted.     The  double 


ENUMERATION    OF   ERYTHROCYTES  279 

rulings  show  when  the  bottom  of  a  column  has  been  reached 
and  also  indicate  the  fourth  column.  In  the  writer's  opin- 
ion it  is  easier  to  count  in  vertical  than  horizontal  rows. 
If  distribution  be  even,  the  difference  between  the  number 
of  cells  in  any  two  such  blocks  should  not  exceed  twenty. 
Instead  of  four  blocks  of  20  squares,  five  blocks  of  16  squares 
may  be  counted,  one  block  in  each  corner  of  the  ruled 
area  and  one  block  in  the  center. 

In  order  to  avoid  confusion  in  counting  cells  which  lie 
upon  the  border-lines,  the  following  rule  is  generally  adopted : 
Corpuscles  which  touch  the  upper  and  left  sides  should  be 
counted  as  if  within  the  squares,  those  touching  the  lower  and 
right  sides,  as  outside;  or  vice  versa. 

Diluting  Fluids. — The  most  widely  used  are  Hayem's  and 
Toisson's.  Both  of  these  have  high  specific  gravities,  so 
that,  when  well  mixed,  the  corpuscles  do  not  separate 
quickly.  Toisson's  fluid  is  perhaps  the  better  for  begin- 
ners, because  it  is  colored  and  can  easily  be  seen  as  it  is 
drawn  into  the  pipet.  It  stains  the  nuclei  of  leukocytes 
blue,  but  this  is  no  real  advantage.  It  must  be  filtered 
frequently  because  of  the  ready  growth  of  fungi  in  it. 
Hayem's  fluid  is  to  be  preferred  for  routine  work.  For 
convenience  in  filling  pipets  the  fluids  should  be  kept  in 
small  wide-mouth  bottles. 

Hayem's  Fluid  Toisson's  Fluid 

Mercuric  chlorid 0.5         Sodium  chlorid i  .0 

Sodium  sulphate 5.0         Sodium  sulphate 8.0 

Sodium  chlorid 1.0         Glycerin 30 .  o 

Distilled  water 200.0         Distilled  water 160.0 

Methyl-violet,    5    B    to   give   a 
strong  purple  color. 

Sources  of  Error. — The  most  common  sources  of  error  in 
making  a  blood-count  are: 

(a)  Inaccurate    dilution,    usually    from    faulty    technic. 


28o  THE   BLOOD 

occasionally  from  inaccurately  graduated  pipets.     Only  an 
instrument  of  standard  make  can  be  relied  upon. 

(b)  Too  slow  manipulation,  allowing  a  little  of  the  blood 
to  coagulate  and  remain  in  the  capillary  portion  of  the 
pipet. 

(c)  Inaccuracy  in  depth  of  counting  chamber  usually  due 
to  imperfect  application  of  the  cover-glass,  but  sometimes  to 
faulty  manufacture  or  to  softening  of  the  cement  by  alco- 
hol or  heat.  The  slide  should  not  be  cleaned  with  alcohol 
nor  left  to  lie  in  the  warm  sunshine. 

(d)  Uneven  distribution  of  the  corpuscles.  This  results 
when  the  blood  has  partially  coagulated,  when  it  is  not 
thoroughly  mixed  with  the  diluting  fluid,  or  when  the  cover- 
glass  is  not  applied  soon  enough  after  the  drop  is  placed 
upon  the  disk. 

(e)  The  presence  of  yeasts,  which  may  be  mistaken  for 
corpuscles,  in  the  diluting  fluid. 

Cleaning  the  Instrument. — The  instrument  should  be 
cleaned  immediately  after  using,  and  the  counting  chamber 
and  cover  must  be  cleaned  again  just  before  use. 

Transfer  the  rubber  tube  to  the  small  end  of  the  pipet  and 
draw  through  it,  successively,  water,  alcohol,  ether,  and  air. 
This  can  be  done  with  the  mouth,  but  it  is  much  better  to 
use  a  rubber  bulb  or  suction  filter  pump.  When  the  mouth 
is  used,  the  moisture  of  the  breath  will  condense  upon  the 
interior  of  the  pipet  unless  the  fluids  be  shaken,  and  not 
blown,  out.  If  blood  has  coagulated  in  the  pipet — which 
happens  when  the  work  is  done  too  slowly — ^dislodge  the 
clot  with  a  horsehair,  never  with  a  wire,  and  clean  with 
strong  sulphuric  acid,  or  let  the  pipet  stand  over  night  in  a 
test-tube  of  the  acid.  Even  if  the  pipet  does  not  become 
clogged,  it  should  be  occasionally  cleaned  in  this  way. 
When  the  etched  graduations  on  the  pipets  become  dim, 
they  can  be  renewed  by  rubbing  with  a  wax  pencil. 

Wash  the  counting-chamber  and  the  cover  with  water  and 


ENUMERATION    OF   ERYTHROCYTES 


281 


dry  them  with  clean  soft  Hnen.  Alcohol  may  be  used  to 
clean  the  latter,  but  never  the  former,  although  a  handker- 
chief slightly  moistened  with  alcohol  may  be  used  to  wipe 
off  the  surface  of  the  ruled  disk  and  the  platform. 

Burker's  hemacytometer  (Fig.  102). — This  modification 
of  the  Thoma-Zeiss  instrument  allows  of  greater  accuracy. 


Fig.  102. — Barker's  hemacytometer  with  pipets  for  counting  red- 
corpuscles:  A,  counting  slide  with  cover-glass  in  place;  B,  pipet  for 
measuring  blood;  C,  pipet  for  measuring  diluting  fluid;  D,  pipet  for 
transferring  diluted  blood  to  slide;  E,  mixing  flask.  In  place  of  the 
flask  and  pipets  here  shown  the  instrument  is  now  generally  supplied 
with  the  regular  Thoma  mixing  pipets. 


Originally  it  consisted  of  a  counting  slide  with  cover-glass, 
three  pipets — for  (a)  measuring  blood,  {b)  measuring 
diluting  fluid,  and  (c)  transferring  the  diluted  blood  to  the 
slide — and  one  or  more  small  flasks  for  mi.xing  blood  and 
diluting  fluid.  As  usually  sold  at  present  it  consists 
simply  of  the  Biirker  counting  slide  and  the  regular  Thoma 
mixing  pipets. 


282  THE   BLOOD 

The  floor-piece  of  the  counting  slide,  instead  of  being  cir- 
cular, as  in  the  Thoma-Zeiss  instrument,  consists  of  a  plate 
of  glass  5  mm.  wide  and  25  mm.  long,  which  extends  across 
the  slide.  This  is  divided  across  the  middle  by  a  deep 
groove  1.5  mm.  wide,  and  upon  each  portion  is  a  ruled  area. 
On  each  side  of  the  floor-piece  and  separated  from  it  by  a 
ditch  is  a  glass  platform  o.i  mm.  higher  than  the  ruled 
areas.  When  the  cover-glass  is  adjusted  upon  the  platform, 
the  ends  of  the  floor-piece  project  beyond  it.  There  are 
two  types  of  the  counting  slide:  one  with  cover-glass  clamps, 
as  shown  in  Fig.  102;  one  lacking  them.  The  clamps  are 
by  no  means  necessary  but  are  a  decided  advantage. 

When  the  count  is  to  be  made,  the  cover-glass  is  carefully 
adjusted  so  as  to  show  Newton's  bands,  and  is  clamped  in 
place  if  the  counting  slide  is  supplied  with  clamps.  A  drop 
of  the  diluted  blood  is  then  placed  on  each  of  the  projecting 
ends  of  the  floor-piece.  The  fluid  will  run  under  the  cover 
by  capillary  attraction.  Care  must  be  exercised  to  use 
just  enough  fluid  to  fill  the  space  between  the  cover 
and  the  floor-piece.  The  slide  is  now  placed  on  the 
microscope  with  the  diaphragm  wide  open  and  viewed 
obliquely  with  the  unaided  eye.  If  the  film  of  corpuscles  as 
seen  in  this  way  is  not  uniform,  the  slide  must  be  cleaned 
and  filled  again.  The  count  is  made  in  the  usual  way.  As 
originally  supplied  the  instrument  had  a  special  ruling,  but 
the  Neubauer  ruling  is  now  generally  used. 

Since  this  counting  chamber  has  two  independent  rul- 
ings it  can  be  filled  for  both  the  red  and  white  counts  at  the 
same  time. 

Levy  Counting  Chamber  (Fig.  103). — In  this  new  Ameri- 
can-made counting  slide  the  Biirker  principle  is  utilized  but 
the  construction  is  somewhat  different  and  apparently 
more  substantial.  The  slide  has  a  matte  surface  which 
makes  it  impossible  to  bring  out  Newton's  bands  but 
which  has  certain  compensating  advantages.     The  makers 


ENUMERATION   OF   ERYTHROCYTES 


283 


state  that  it  will  soon  be  obtainable  with  cover-glass 
clamps  which  will  be  a  decided  improvement.  The  ordinary 
Thoma  mixing  pipets  are  supplied  with  it. 

Thoma-Metz  Hemacytometer  (Fig.  104). — This  new  in- 
strument introduces  certain  conveniences  into  the  routine 
counting  of  both  red  cells  and  leukocytes.     Its  special 


Y 

.^gSqanm. 
^mm.deep. 

._, 

J 



MADE     BV 

PHILADELPHIA.PA. 

o.s>- 

ArtKTirl{Th!.!n?.3  Cj. 

Pig.   103. — Levy's     counting     chamber,     Burker     type. 

feature  is  that  the  ruling  is  engraved  upon  a  disk  in  the 
ocular  instead  of  upon  the  counting  slide.  This  disk  is 
ruled  with  a  large  circle  and  with  a  square,  which  in  turn  is 
subdivided  into  four  smaller  squares. 

For  the  red  count  the  squares  are  used.     The  four  small 
squares  have  each  the  same  value  as  the  small  squares  of  the 


© 


Fig.  104. — Thoma-Metz  hemacytometer  and  diagram  showing  ruling 

in  ocular. 

Thoma  ruling  (3^oo  sq.  mm.),  and  the  count  may  be  con- 
ducted as  already  described. 

The  circle  is  used  for  counting  leukocytes.  Its  area  cor- 
responds to  0.1  sq.  mm.  when  the  correct  magnification  is 
used.  The  leukocytes  are  counted  as  in  the  author's  circle 
method  described  on  page  298. 


284  "     THE  BLOOD 

A  decided  advantage  of  this  instrument  is  that  the  ruled 
lines  are  always  sharp  and  clear.  The  eye-lens  of  the  ocular 
can  be  focused  to  suit  different  eyes.  The  chief  disadvan- 
tage and  a  source  of  inaccuracy  lies  in  the  fact  that  the  values 
of  the  ruled  areas  vary  according  to  magnification.  The 
makers  say  that  values  are  correct  as  above  given  when  the 
Leitz  No.  6  objective  (4-mm.  focus)  is  used  with  tube  length 
of  170  mm.  Slight  variations  with  other  objectives  can  be 
compensated  by  altering  the  tube  length.  For  accurate 
evaluation  a  square  is  ruled  on  the  counting  slide,  and  the 
tube  length  should  be  so  adjusted  that  this  square  exactly 
coincides  with  the  large  square  in  the  ocular. 

V.  COLOR  INDEX 

This  is  an  expression  which  indicates  the  amount  of 
hemoglobin  in  each  red  corpuscle  compared  with  the 
normal  amount.  For  example,  a  color  index  of  i.o  in- 
dicates that  each  corpuscle  contains  the  normal  amount 
of  hemoglobin;  of  0.5,  that  each  contains  one-half  the 
normal. 

The  color  index  is  most  significant  in  chlorosis  and 
pernicious  anemia.  In  the  former  it  is  usually  much 
decreased;  in  the  latter,  generally  much  increased.  In 
symptomatic  anemia  it  is  moderately  diminished. 

To  obtain  the  color  index,  divide  the  percentage  of  hemo- 
globin by  the  percentage  of  corpuscles.  The  percentage  of 
corpuscles  is  found  by  multiplying  the  first  two  figures  of  the 
red  corpuscle  count  by  2.  This  simple  method  holds  good 
for  all  counts  of  1,000,000  or  more.  Thus,  a  count  of 
2,500,000  is  50  per  cent,  of  the  normal.  If,  then,  the  hemo- 
globin has  been  estimated  at  40  per  cent.,  divide  40  (the 
percentage  of  hemoglobin)  by  50  the  percentage  of  cor- 
puscles).    This  gives  ^^,  or  0.8,  as  the  color  index. 


VOLUME   INDEX  285 

From  what  has  already  been  said  regarding  the  varia- 
tions in  hemoglobin-instruments,  and  of  the  impossi- 
bility of  fixing  a  normal  standard  for  either  red  cells 
or  hemoglobin  which  is  applicable  to  all  ages  and  in  all 
localities,  it  would  appear  that  color-index  calculations, 
as  above  described,  have  little  value.  Certainly  only 
marked  variations  can  be  considered  in  diagnosis 
unless  one  takes  into  account  the  age  and  sex  of  the 
patient,  the  locality,  and  the  hemoglobin-instrument 
used.  It  has  been  suggested  that  the  index  be  worked 
out  upon  a  basis  of  grams  of  hemoglobin  per  1,000,000 
red  cells,  which  would  make  it  of  real  value,  but  this 
is  not  yet  in  vogue. 

VI.    VOLUME  INDEX 

The  term  "volume  index"  was  introduced  by  Capps 
to  express  the  average  size  of  the  red  cells  of  an  indi- 
vidual compared  with  their  normal  size.  It  is  the  quo- 
tient obtained  by  dividing  the  volume  of  red  corpuscles 
(expressed  in  percentage  of  the  normal)  by  the  number  oi 
red  corpuscles,  also  expressed  in  percentage  of  the  normal. 

The  volume  index  more  or  less  closely  parallels  the 
color  index,  and  variations  have  much  the  same  sig- 
nificance. The  following  are  averages  of  the  examina- 
tions reported  by  Larrabee  in  the  "Journal  of  Medical 
Research:" 

Red  corpuscles    Hemoglobin  per 

per  cubic  cent,  by  Sahli     Color    Volume 

millimeter  instrument        index      index 

Normal  males 5,267,250  103.0  0.98  1.C07 

Normal   females 4,968,667  106.0  1.06  i.coi 

Primary  pernicious  anemia  1,712,166  50.0  1.47  1.270 

Secondary   anemia 3>737ii6o  61.0  0.81  c.790 

Chlorosis 3,205,000  34.5  0.55  0.695 


286  THE  BLOOD 

Method. — The  red  cells  are  counted  and  the  percentage  of 
red  cells  calculated  as  for  the  color  index. 

The  volume  percentage  is  obtained  with  the  hematocrit 
as  follows:  Fill  the  hematocrit  tubes  (Fig.  105)  with  blood, 
and  before  coagulation  takes  place  insert  them  in  the  frame 
and  centrifugalize  for  three  minutes  at  about  8000  to  10,000 
revolutions  a  minute.  The  red  cells  collect  at  the  bottom 
and,  normally,  make  up  one-half  of  the  total  column  of 
blood.  Multiply  the  height  of  the  layer  of  red  cells  (as 
indicated  by  the  graduations  upon  the  side  of  the  tube)  by 
2  to  obtain  the  volume  percentage.  When  the  examination 
cannot  be  made  immediately  after  the  blood  is  obtained, 
the  method  of  Larrabee  is  available.     This  consists  in  mix- 


f   |iiii|iiii|iiii|iiii|iili|im|iiil|iiii|iiii| iiiij|iNiiiiii|'ij|ii.i|i .^ 

\    10  CO  oo»t»  jooo'X)  00  9e^ 


Fig.  105. — Daland  hematocrit  for  use  with  the  centrifuge. 

ing  a  trace  of  sodium  oxalate  with  a  few  drops  of  blood  to 
prevent  coagulation,  drawing  this  mixture  into  a  heavy- 
walled  tube  of  about  2-mm.  caliber,  closing  the  ends  with  a 
rubber  band,  and  waiting  until  sedimentation  is  complete — ■ 
usually  about  three  days.  The  height  of  the  column  is 
then  measured  with  a  millimeter  scale  and  the  percentage 
relation  to  the  normal  calculated. 

After  the  volume  of  the  red  cells  and  the  red  corpuscle 
count  are  thus  expressed  in  percentages,  divide  the  former 
by  the  latter  to  find  the  volume  index.  Example :  Suppose 
the  volume  percentage  is  80  (the  reds  reaching  to  mark  40  on 
hematocrit  tube)  and  that  the  red  count  is  50  per  cent,  of 
the  normal  (2,500,000  per  cubic  millimeter),  then  ^%o> 
or  1.6,  is  the  volume  index. 


ENUMERATION   OF    LEUKOCYTES  287 

VII.   ENUMERATION  OF  LEUKOCYTES 

The  normal  number  of  leukocytes  varies  from  5000  to 
10,000  per  cubic  millimeter  of  blood.  The  number  is 
larger  in  robust  individuals  than  in  poorly  nourished 
ones,  and,  if  disease  be  excluded,  may  be  taken  as  a 
rough  index  of  the  individual's  nutrition.  Since  it  is 
well  to  have  a  definite  standard,  7500  is  generally  adopted 
as  the  normal  for  the  adult.  With  children  the  number 
is  somewhat  greater,  averaging  about  10,000  to  12,000 
in  infants  and  somewhat  below  10,000  in  older  children. 

Decrease  in  Number  of  Leukocytes 

Decrease  in  number  of  leukocytes,  or  leukopenia,  is  not 
important.  It  is  common  in  persons  who  are  poorly 
nourished,  although  not  actually  sick.  The  infectious 
diseases  in  which  leukocytosis  is  absent  (see  p.  291) 
often  cause  a  slight  decrease  of  leukocytes.  Chlorosis 
may  produce  leukopenia,  as  also  pernicious  anemia, 
which  usually  gives  it  in  contrast  to  the  secondary 
anemias,  which  are  frequently  accompanied  by  leukocy- 
tosis. Leukocyte  counts  are,  therefore,  of  some  aid  in 
the  difTerential  diagnosis  of  these  conditions. 

Increase  in  Number  of  Leukocytes 

Increase  in  number  of  leukocytes  is  common  and  of 
great  importance.  It  may  be  considered  under  two 
heads: 

A.  Increase  of  leukocytes  due  to  chemotaxis  and 
stimulation  of  the  blood-making  organs,  or  leukocytosis. 
The  increase  affects  one  or  more  of  the  normal  varieties. 

B.  Increase  of  leukocytes  due  to  leukemia.     Normal 


288  THE  BLOOD 

varieties  are  increased,  but  the  characteristic  feature  is 
the  appearance  of  great  numbers  of  abnormal  cells. 

The  former  may  be  classed  as  a  transient,  the  latter,  as 
a  permanent,  increase. 

A.  Leukocytosis 

This  term  is  variously  used.  By  some  it  is  applied  to 
any  increase  in  number  of  leukocytes;  by  others  it  is 
restricted  to  increase  of  the  polymorphonuclear  neutro- 
philic variety.  As  has  been  indicated,  it  is  here  taken 
to  mean  a  transient  increase  in  number  of  leukocytes, 
that  is,  one  caused  by  chemotaxis  and  stimulation  of  the 
blood-producing  structures,  in  contrast  to  the  permanent 
increase  caused  by  leukemia. 

By  chemotaxis  is  meant  that  property  of  certain  agents 
by  which  they  attract  or  repel  living  cells — positive 
chemotaxis  and  negative  chemotaxis  respectively.  An 
excellent  illustration  is  the  accumulation  of  leukocytes 
at  the  site  of  inflammation,  owing  to  the  positively 
chemotactic  influence  of  bacteria  and  their  products. 
A  great  many  agents  possess  the  power  of  attracting 
leukocytes  into  the  general  circulation.  Among  these 
are  many  bacteria  and  certain  organic  and  inorganic 
poisons. 

Chemotaxis  alone  will  not  explain  the  continuance  of 
leukocytosis  for  more  than  a  short  time.  It  is  probable 
that  substances  which  are  positively  chemotactic  also 
stimulate  the  blood-producing  organs  to  increased  forma- 
tion of  leukocytes;  and  in  at  least  one  form  of  leukocy- 
tosis such  stimulation  apparently  plays  the  chief  part. 

As  will  be  seen  later,  there  are  several  varieties  of  leu- 
kocytes in  normal  blood,  and  most  chemotactic  agents 


ENUMERATION    OF   LEUKOCYTES  289 

attract  only  one  variety,  and  either  repel  or  do  not  in- 
fluence the  others.  It  practically  never  happens  that 
all  are  increased  in  the  same  proportion.  The  most 
satisfactory  classification  of  leukocytoses  is,  therefore, 
based  upon  the  type  of  leukocyte  chiefly  affected. 

Theoretically,  there  should  be  a  subdivision  for  each 
variety  of  leukocyte,  e.g.,  polymorphonuclear  leuko- 
cytosis, lymphocytic  leukocytosis,  eosinophilic  leuko- 
cytosis, large  mononuclear  leukocytosis,  etc.  Practi- 
cally, however,  only  two  of  these,  polymorphonuclear 
leukocytosis  and  lymphocytic  leukocytosis,  need  be  con- 
sidered under  the  head  of  Leukocytosis.  Increase  in 
number  of  the  other  leukocytes  will  be  considered 
when  the  individual  cells  are  described  (see pp.  326-339). 
They  are  present  in  the  blood  in  such  small  numbers 
normally  that  even  a  marked  increase  scarcely  affects 
the  total  leukocyte  count;  and,  besides,  substances 
which  attract  them  into  the  circulation  frequently  repel 
the  polymorphonuclears,  so  that  the  total  number  of 
leukocytes  may  actually  be  decreased. 

The  polymorphonuclear  neutrophiles  are  capable  of 
active  ameboid  motion,  and  are  by  far  the  most  numer- 
ous of  the  leukocytes.  Lymphocytes  are  about  one- 
third  as  numerous  and  have  little  independent  motion. 
As  one  would,  therefore,  expect,  marked  differences 
exist  between  the  two  types  of  leukocytosis:  polynuclear 
leukocytosis  is  more  or  less  acute,  coming  on  quickly  and 
often  reaching  high  degree;  whereas  lymphocytic  leuko- 
cytosis is  more  chronic,  comes  on  more  slowly,  and  is 
never  so  marked. 

1.  Polymorphonuclear  Neutrophilic  Leukocy- 
tosis.— Polymorphonuclear  leukocytosis  may  be  either 

19 


290  THE  BLOOD 

physiologic  or  pathologic.  A  count  of  20,000  would  be 
considered  a  marked  leukocytosis;  of  30,000,  high; 
above  50,000,  extremely  high. 

(i)  Physiologic  Polymorphonuclear  Leukocytosis. — 
This  is  never  very  marked,  the  count  seldom  exceeding 
12,000  per  cubic  millimeter.  It  may  occur:  (a)  In  the 
new-born;  (b)  in  pregnancy;  (c)  during  digestion;  and 
(d)  after  cold  baths.  There  is  moderate  leukocytosis  in 
the  moribund  state :  this  is  commonly  classed  as  physio- 
logic, but  is  probably  due  mainly  to  terminal  infection. 

The  increase  in  these  conditions  is  not  limited  to  the 
polymorphonuclears.  Lymphocytes  are  likewise  in- 
creased in  varying  degrees,  most  markedly  in  the 
new-born. 

In  view  of  the  leukocytosis  of  digestion,  which 
usually  increases  the  leukocytes  by  about  30  per  cent., 
the  hour  at  which  a  leukocyte  count  is  made  should 
always  be  recorded.  Digestive  leukocytosis  is  most 
marked  three  to  five  hours  after  a  hearty  meal  rich  in 
protein,  especially  when  such  a  meal  follows  a  long  fast. 
It  is  absent  in  pregnancy  and  when  leukocytosis  from 
any  other  cause  exists.  It  is  usually  absent  in  cancer  of 
the  stomach,  a  fact  which  may  be  of  some  help  in  the 
diagnosis  of  this  condition,  but  repeated  examinations 
and  careful  technic  are  essential. 

(2)  Pathologic  Polymorphonuclear  Leukocytosis. — 
In  general,  the  response  of  the  leukocytes  to  chemotaxis 
is  a  conservative  process.  It  has  been  compared  to  the 
gathering  of  soldiers  to  destroy  an  invader.  This  is 
accomplished  partly  by  means  of  phagocytosis — actual 
ingestion  of  the  enemy — and  partly  by  means  of  chemic 
substances  which  the  leukocytes  produce. 


ENUMERATION   OF   LEUKOCYTES  29I 

In  those  diseases  in  which  leukocytosis  Is  the  rule  the 
degree  of  leukocytosis  depends  upon  two  factors:  the 
severity  of  the  infection  and  the  resistance  of  the  individual. 
A  well-marked  leukocytosis  usually  indicates  good  resist- 
ance. A  mild  degree  means  that  the  body  is  not  react- 
ing well,  or  else  that  the  infection  is  too  slight  to  call 
forth  much  resistance.  Leukocytosis  may  be  absent 
altogether  when  the  infection  is  extremely  mild,  or  when 
it  is  so  severe  as  to  overwhelm  the  organism  before  it  can 
react.  When  leukocytosis  is  marked,  a  sudden  fall  in 
the  count  may  be  the  first  warning  of  a  fatal  issue. 
These  facts  are  especially  true  of  pneumonia,  diphtheria, 
and  abdominal  inflammations,  in  which  conditions  the 
degree  of  leukocytosis  is  of  considerable  prognostic  value. 

The  classification  here  given  follows  Cabot: 

(a)  Infectious  and  Inflammatory.— The  majority  of 
infectious  diseases  produce  leukocytosis.  The  most 
notable  exceptions  are  influenza,  measles,  German 
measles,  tuberculosis,  except  when  invading  the 
meninges  or  when  complicated  by  mixed  infection, 
and  typhoid  fever,  in  which  leukocytosis  indicates  an 
inflammatory  complication. 

All  inflammatory  and  suppurative  diseases  cause  leu- 
kocytosis, except  when  slight  or  well  walled  off.  Appen- 
dicitis has  been  studied  with  especial  care  in  this  connec- 
tion, and  the  conclusions  now  generally  accepted  proba- 
bly hold  good  for  most  acute  intra-abdominal  inflam- 
mations. A  marked  leukocytosis  (20,000  or  more) 
nearly  always  indicates  abscess,  peritonitis,  or  gan- 
grene, even  though  the  clinical  signs  be  slight.  Absence 
of  or  mild  leukocytosis  indicates  a  mild  process,  or  else 
an  overwhelmingly  severe  one ;  and  operation  may  safely 


292  THE  BLOOD 

be  postponed  unless  the  abdominal  signs  are  very  marked. 
On  the  other  hand,  no  matter  how  low  the  count,  an 
increasing  leukocytosis — counts  being  made  hourly 
• — indicates  a  spreading  process  and  demands  operation, 
regardless  of  other  symptoms. 

Leukocyte  counts  alone  are  often  disappointing,  hut  are 
of  much  more  value  when  considered  in  connection  with 
a  differential  count  of  polymorphonuclears  (see  p.  331). 

(h)  Malignant  Disease. — Leukocytosis  occurs  in  about 
one-half  of  the  cases  of  malignant  disease.  In  many 
instances  it  is  probably  independent  of  any  secondary 
infection,  since  it  occurs  in  both  ulcerative  and  non- 
ulcerative cases.  It  seems  to  be  more  common  in  sar- 
coma than  in  carcinoma.  Very  large  counts  are  rarely 
noted. 

(c)  Posthemorrhagic. — Moderate  leukocytosis  follows 
hemorrhage  and  disappears  in  a  few  days.  In  cases 
of  ruptured  tubal  pregnancy  with  hemorrhage  into 
the  peritoneal  cavity  the  count  usually  reaches  18,000 
to  30,000. 

{d)  Toxic. — This  is  a  rather  obscure  class,  which  in- 
cludes gout,  chronic  nephritis,  acute  yellow  atrophy 
of  the  liver,  ptomain-poisoning,  prolonged  chloroform 
narcosis,  and  quinin-poisoning.  Leukocytosis  may  or 
may  not  occur  in  these  conditions,  and  is  not  important. 

{e)  Drugs. — This  also  is  an  unimportant  class.  Most 
tonics  and  stomachics  and  many  other  drugs  produce  a 
slight  leukocytosis.  A  moderate  leukocytosis  may  also 
occur  as  a  result  of  prolonged  ether  anesthesia. 

2.  Lymphocytic  Leukocytosis. — This  is  character- 
ized by  an  increase  in  the  total  leukocyte  count,  accom- 
panied by  an  increase  in  the  percentage  of  lymphocytes. 


ENUMERATION    OF   LEUKOCYTES  293 

The  word  "lymphocytosis"  is  often  used  in  the  same 
sense.  It  is  better,  however,  to  use  the  latter  as  refer- 
ring to  any  increase  in  the  absolute  number  of  lympho- 
cytes, without  regard  to  the  total  count,  since  an  ab- 
solute increase  in  number  of  lymphocytes  is  frequently 
accompanied  by  a  normal  or  subnormal  leukocyte  count, 
owing  to  loss  of  polymorphonuclears. 

Lymphocytic  leukocytosis  is  probably  due  more  to 
stimulation  of  blood-making  organs  than  to  chemotaxis. 
It  is  less  common,  and  is  rarely  so  marked  as  a  poly- 
morphonuclear leukocytosis.  When  marked,  the  blood 
cannot  be  distinguished  from  that  of  lymphatic  leukemia. 

A  marked  lymphocytic  leukocytosis  occurs  in  per- 
tussis. It  is  said  to  appear  early  in  the  catarrhal  stage 
and  to  reach  its  maximum  at  the  height  of  the  par- 
oxysmal stage,  after  which  it  gradually  subsides.  In 
30  well-marked  cases  studied  by  Schneider  the  average 
leukocyte  count  was  19,000  in  the  first  week,  rising  to 
about  27,000  in  the  third.  His  lowest  counts  in  the 
first  week  were  12,600  and  in  the  third  16,800.  Leuko- 
cyte counts  would  therefore  seem  to  have  great  value 
in  diagnosis,  but  in  our  experience  they  have  often  been 
disappointing  since  in  many  mild  cases  the  count  does 
not  rise  above  what  may  be  regarded  as  a  high  normal 
for  children  before  the  characteristic  whoop  begins. 
There  is  moderate  lymphocytic  leukocytosis  in  other 
diseases  of  childhood,  as  rickets,  scurvy,  and  especially 
hereditary  syphilis,  where  the  blood  picture  may  ap- 
proach that  of  pertussis.  It  must  be  borne  in  mind 
in  this  connection  that  lymphocytes  are  normally  more 
abundant  in  the  blood  of  children  than  in  that  of 
adults. 


294 


THE  BLOOD 


Slight  lymphocytic  leukocytosis  occurs  in  many  other 
pathologic  conditions,  but  is  of  little  significance. 

B.  Leukemia 

This  is  an  idiopathic  disease  of  the  blood  making 
organs,  which  is  accompanied  by  an  enormous  increase 
in  number  of  leukocytes.  The  leukocyte  count  some- 
times reaches  1,000,000  per  cubic  millimeter,  and  leu- 
kemia is  always  to  be  suspected  when  it  exceeds  50,000. 
Lower  counts  do  not,  however,  exclude  it.  The  subject 
is  more  fully  discussed  later  (see  p.  387). 


r 

IRnin 

Iiiii 
iiiiii 
iiiii 

11 ! 

ill 

ii  ai       i|| 

r 

1 

1 

iiiii 
iiiii 

Sliiii  ii 
II""'" ' 

Fig.  106. — Tiirck  ruling  for  counting  chamber  (  X  IS). 

Method  of  Counting  Leukocytes 

The  leukocytes  may  be  counted  with  any  one  of  the 
hemacytometers  already  described  (see  pp.  273-284). 
Numerous  modifications  of  the  original  ruling  have 
been  introduced,  notably  the  Tiirck,  the  Zappert-Ewing, 
and  the  Neubauer  (Figs.  106,  107,  108),  which  give 
a  ruled  area  of  9  sq.  mm.,  the  center  having  the  Thoma 


ENUMERATION    OF   LEUKOCYTES 


295 


ruling.     Of   these   the    Neubauer  may  be   especially 
commended.     Some  of  them  were  originally  devised 


B.^,L.0.C9 


Fig.  107. — Zappert-Ewing  ruling  for  counting  chamber  (  X  IS)- 


■■■■ 
■■■■ 

■III 

SB 

BIB 

■■  ■!!!! 

mill 

lilllHi 

llliil 
III  ill 

In 

1                                                 B.aL.O.C9 

Fig.  108.- — Neubauer  ruling  for  counting  chamber  (X  iS)- 

for  counting  the  leukocytes  in  the  same  dilution  with 
the  red  corpuscles.     The  two  kinds  of  cell  are  easily 


296 


THE   IBLOOD 


distinguished,  especially  when  Toisson's  diluting  fluid 
is  used.  The  red  cells  are  counted  in  the  central  por- 
tion in  the  usual  manner,  after  which  all  the  leukocytes 
in  the  whole  area  of  9  sq.  mm.  are  counted;  and  the 
number  in  a  cubic  millimeter  of  undiluted  blood  is 
then  calculated.  Bass'  new  ruling  (Fig.  109)  covers 
4  sq.  mm.  and  is  used  in  a  similar  manner.  With  the 
older  Thoma  ruling  the  reds  and  the  leukocytes  may 
be  counted  in  the  same  preparation  by  adjusting  the 


Fig.   109. — Bass  ruling  for  counting  chamber  (X  is). 


microscopic  field  to  a  definite  size,  and  counting  a  suffi- 
cient number  of  fields,  as  described  later. 

Although  less  convenient,  it  is  more  accurate  to  count 
the  leukocytes  separately,  with  less  dilution  of  the 
blood,  as  follows: 

Technic. — A  larger  drop  of  blood  is  required  than  for 
counting  the  erythrocytes,  and  more  care  in  filling  the  pipet, 
since  the  bore  is  considerably  larger  than  that  of  the 
"red"  pipet.  Boggs  has  suggested  a  device  (Fig.  no) 
which  enables  one  to  draw  in  the  blood  more  slowly  and 
hence  more  accurately.  He  cuts  the  rubber  tube  and  inserts 
a  Wright  "throttle."     This  consists  of  a  section  of  glass 


ENUMERATION    OF    LEUKOCYTES 


297 


tubing  within  which  a  capillary  tube  drawn  out  to  a  fine 
thread  is  cemented  with  sealing  wax.  After  sealing  in 
place  the  tip  is  broken  off  with  forceps,  so  that  upon 
gentle  suction  it  will  just  allow  air  to  pass. 

Use  the  pipet  with  1 1  engraved  above  the  bulb.  ^  Suck  the 
blood  to  the  mark  0.5  or  i.o,  and  the  diluting  fluid  to  the 
mark  11.     This  gives  a  dilution  of  i :  20  or  i :  10,  respectively. 


Fig.  1 10. — 


no. — iJoggs'   "throttle  control"  for  blood-counting  pipet,   and 
enlarged  diagram  showing  construction  of  the  throttle. 


The  dilution  of  i :  20  is  easier  to  make.  Mix  well  by  shaking 
in  all  directions  except  in  the  long  axis  of  the  pipet;  blow  out 
2  or  3  drops,  place  a  drop  in  the  counting  chamber,  and 
adjust  the  cover  as  already  described  (see  pp.  276,  277). 

Examine  with  a  low  power  to  see  that  the  cells  are  evenly 
distributed.     Count  with  the  i6-mm.  objective  and  a  high 

^  In  some  cases  of  leukemia  with  very  high  count  it  may  be  neces- 
sary to  use  the  "red"  pipet  with  dilution  of  i:  loo 


298  THE  BLOOD 

eye-piece,  or  with  the  long-focus  4  mm.  and  a  low  eye-piece. 
An  8-mm.  objective  will  be  found  very  satisfactory  for  this 
purpose.  As  one  gains  experience  one  will  rely  more  upon 
the  lower  powers. 

With  the  Thoma  ruling  count  all  the  leukocytes  in  the 
square  millimeter,  multiply  by  10  to  find  the  number  in 
I  cu.  mm.  of  diluted  blood,  and  by  the  dilution  to  find  the 
number  per  cubic  millimeter  of  undiluted  blood.  In  every 
case  at  least  200  leukocytes  must  be  counted  as  a  basis  for 
calculation,  and  it  is  much  better  to  count  500.  This  will 
necessitate  examination  of  several  drops  from  the  pipet. 
With  the  rulings  which  cover  9  sq.  mm.  a  sufficient  number 
can  usually  be  counted  in  one  drop,  but  the  opportunity 
for  error  is  very  much  greater  when  only  one  drop  is 
examined. 

In  routine  work  the  author's  modification  of  the  "circle" 
method  is  very  satisfactory.  It  requires  a  4-mm.  objective, 
and  is,  therefore,  especially  desirable  for  beginners,  who  are 
usually  unable  accurately  to  identify  leukocytes  with  a  lower 
power.  The  student  is  frequently  confused  by  particles  of 
dirt,  remains  of  red  cells,  and  yeast  cells  which  are  prone  to 
grow  in  the  diluting  fluid.  Draw  out  the  sliding  tube  of  the 
microscope  until  the  field  of  vision  is  such  as  shown  in  Fig. 
III.  One  side  of  the  field  is  tangent  to  one  of  the  ruled  lines, 
A,  while  the  opposite  side  just  cuts  the  corners,  B  and  C,  of 
the  seventh  squares  in  the  rows  above  and  below  the  dia- 
meter line.  When  once  adjusted,  a  scratch  is  made  upon 
the  draw-tube,  so  that  for  future  counts  the  tube  has  only 
to  be  drawn  out  to  the  mark.  The  area  of  this  microscopic 
field  is  0.1  sq.  mm.  With  a  dilution  of  1:20,  count  the 
leukocytes  in  20  such  fields  upon  diflferent  parts  of  the  disk 
without  regard  to  the  ruled  lines,  and  to  their  sum  add  two 
ciphers.  With  dilution  of  i :  10,  count  10  such  fields,  and 
add  two  ciphers.  Thus,  with  1:10  dilution,  if  1 50  leukocytes 
were  counted  in  10  fields,  the  leukocyte  count  would  be 


ENUMERATION   OF    LEUKOCYTES 


299 


15,000  per  cubic  millimeter.  To  compensate  for  possible 
unevenness  of  distribution,  it  is  best  to  count  a  row  of  fields 
horizontally  and  a  row  vertically  across  the  disk.  This 
method  is  applicable  to  any  degree  of  dilution  of  the  blood, 
and  is  simple  to  remember:  one  always  counts  a  number  of 


1           1 

1               1 

-'T 1" 

.-4- L.__ 

1              1 

1              1 
,              1 

--r----t- 

1           1 

1   : 





r 

1     1          1 

1     1          1 

r-T-t r-- 

L  .  -^ 

r    '    1 

'          '     ' 

y'^ 

N 

r       r       T-                r      - 

■    T-t 1-- 

a       '     -4-        -       i 

/ 

\ 

[         / 

1                        /    L 

:  1     1 

-f--i V- 

1                              1     \ 

/ 
/ 

1                              1                     \ 
1                             1 

--+ f 1 

1                             1 
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1            1 

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1           1 

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--  \ 

-  ^ 

H 



—  -J 

— 

1     1     1         '    . 
1     '     1         1 

1,1      ' 
1  >  1      ' 

Fig.   III. 


Size  of  field  required  in  counting  leukocytes  as  described  in 
the  text. 


f.elds  equal  to  the  number  of  times  the  blood  has  been  diluted, 
and  adds  two  ciphers.  Evidence  of  the  convenience  of  using 
a  circle  of  this  size  is  afforded  by  its  adoption  in  the  new 
Thoma-Metz  instrument. 

It  is  sometimes  impossible  to  obtain  the  proper  size  of 


300  THE   BLOOD 

field  with  the  objectives  and  eye-pieces  at  hand.  In  such 
case  place  a  cardboard  or  stiff  paper  disk  with  a  circular 
opening  upon  the  diaphragm  of  the  eye-piece,  and  adjust 
the  size  of  the  field  by  drawing  out  the  tube.  The  circular 
opening  can  be  cut  with  a  sharp  cork-borer. 

Diluting  Fluids. — The  diluting  fluid  should  dissolve  the 
red  corpuscles  so  that  they  will  not  obscure  the  leukocytes. 
The  simplest  fluid  is  a  i  per  cent,  solution  of  acetic  acid. 
More  satisfactory  is  the  following:  glacial  acetic  acid,  i  c.c; 
I  per  cent,  aqueous  solution  of  gentian- violet,  i  c.c;  distilled 
water,  loo  c.c.  These  solutions  must  be  filtered  frequently 
to  remove  yeasts  and  molds. 

Vm.  ENXJMERATION  OF  BLOOD-PLATELETS 

The  average  normal  number  of  platelets  is  variously 
given  as  200,000  to  700,000  per  cubic  millimeter  of 
blood.  Many  of  the  counts  were  obtained  by  workers 
who  used  inaccurate  methods.  Using  their  own  reliable 
method,  Wright  and  Kinnicutt  found  the  normal  average 
to  range  from  263,000  to  336,000.  Physiologic  varia- 
tions are  marked;  thus,  the  number  increases  as  one 
ascends  to  a  higher  altitude,  and  is  higher  in  winter  than 
in  summer.  There  are  unexplained  variations  from  day 
to  day;  hence  a  single  abnormal  count  should  not  be 
taken  to  indicate  a  pathologic  condition. 

Pathologic  variations  are  often  very  great.  Owing 
to  lack  of  knowledge  as  to  the  function  of  the  platelets 
and  to  the  earlier  imperfect  methods  of  counting,  the 
clinical  significance  of  these  variations  is  uncertain. 
The  following  facts  seem,  however,  to  be  established: 

(a)  In  acute  infectious  diseases  the  number  is  sub- 
normal or  normal.  If  the  fever  ends  by  crisis,  the  crisis 
is  accompanied  by  a  rapid  and  striking  increase. 


ENUMERATION   OF  BLOOD-PLATELETS  3OI 

(b)  In  secondary  anemia  platelets  are  generally  in- 
creased, although  sometimes  decreased.  In  pernicious 
anemia  they  are  always  greatly  diminished,  and  an 
increase  should  exclude  the  diagnosis  of  this  disease. 

(c)  They  are  decreased  in  chronic  lymphatic  leukemia, 
and  greatly  increased  in  the  myelogenous  form. 

(d)  In  purpura  haemorrhagica  the  number  is  enor- 
mously diminished. 

(e)  The  platelets  are  somewhat  increased  in  tuber- 
culosis. 

Blood-platelets  are  difficult  to  count,  owing  to  the 
rapidity  with  which  they  disintegrate,  and  to  their  great 
tendency  to  adhere  to  any  foreign  body  and  to  each 
other. 

Method  of  Wright  and  Kinnicutt. — This  method  is  simple, 
appears  to  be  accurate,  and  certainly  yields  uniform  results. 

The  platelets  are  counted  with  the  hemacytometer 
already  described,  using  a  dilution  of  i :  100.  The  diluting 
fluid  consists  of  2  parts  of  an  aqueous  solution  of  brilliant 
cresyl  blue  (1:300)  and  3  parts  of  an  aqueous  solution  of 
potassium  cyanid  (1:1400).  These  two  solutions  must  be 
kept  in  separate  bottles  and  mixed  and  filtered  imme- 
diately before  using.  The  cresyl  blue  solution  is  per- 
manent but  molds  have  a  tendency  to  grow  in  it.  The 
cyanid  solution  deteriorates  after  about  ten  days.  Rapid 
work  is  necessary  in  order  to  prevent  clumping  of  the 
platelets.  After  the  blood  is  placed  in  the  counting- 
chamber  it  is  allowed  to  stand  for  ten  minutes  or  longer  in 
order  that  the  platelets  may  settle.  The  count  is  made 
with  a  high  dry  objective  and  a  high  ocular.  The  plate- 
lets appear  as  rounded,  lilac-colored  bodies;  the  reds  are 
decolorized,  appearing  only  as  shadows. 

The  leukocytes  are  stained  and  may  be  counted  at  the 
same  time. 


302  THE  BLOOD 

Ottenberg  and  Rosenthal  have  recently  suggested  3  per 
cent,  sodium  citrate  as  a  diluting  fluid  to  be  used  in  the 
same  manner  as  that  of  Wright  and  Kinnicutt.  This 
may  be  colored  by  adding  0.2  per  cent,  of  brilliant  cresyl 
blue  or  methyl  violet,  but  the  fluid  must  then  be  made 
up  freshly  each  day  since  it  deteriorates  rapidly. 

IX.  STUDY  OF  STAINED  BLOOD 

A.  Making  and  Staining  Blood-films 

!.  Spreading  the  Film. — Thin,  even  films  are  essen- 
tial to  accurate  and  pleasant  work.  They  more  than 
compensate  for  the  time  spent  in  learning  to  make  them. 
.There  are  certain  requisites  for  success  with  any  me- 
thod: (a)  The  slides  and  covers  must  be  perfectly  clean: 
thorough  washing  with  soap  and  water,  rubbing  with 
alcohol  and  drying  on  a  clean  handkerchief  will  usually 
suffice;  {b)  the  drop  of  blood  must  not  be  too  large;  (c) 
the  work  must  be  done  quickly,  before  coagulation 
begins. 

TJie  blood  is  obtained  from  the  finger-tip  or  the  lobe 
of  the  ear,  as  for  a  blood  count;  only  a  very  small  drop  is 
required,  usually  about  the  size  of  a  large  pin-head. 
The  size  of  the  drop  largely  determines  the  thickness 
of  the  film.  The  proper  thickness  will  depend  upon 
the  purpose  for  which  the  film  is  made.  For  the  struc- 
ture of  blood  cells  and  the  malarial  parasite  it  should 
be  so  thin  that,  throughout  the  greater  part  of  the  film, 
the  red  corpuscles  lie  in  a  single  layer,  close  together 
but  not  overlapping.  In  our  class  work  we  insist  that 
all  films  meet  this  requirement.  For  routine  differen- 
tial counting  of  leukocytes  a  film  in  which  the  red  cells 
are  piled  up  somewhat  is  best  because  the  number  of 


STUDY    OF    STAINED    BLOOD 


303 


leukocytes  in  a  given  area  is  thus  greatly  increased  and 
the  tedium  of  counting  is  corresponding  lessened.  The 
film  must  not,  upon  the  other  hand,  be  so  thick  that  iden- 
tification of  the  various  leukocytes  becomes  difficult. 

Nearly  all  ordinary  slides  are  curved.  In  order  that 
they  may  lie  firmly  upon  the  microscope  stage  without 
rocking,  the  blood  film  should  be  spread  upon  the  con- 
vex side,  which  is  recognized  by  laying  the  slide  flat 
upon  the  table  and  twirling  it  rapidly  by  snapping  the 


Fig.  112. — Spreading  the  film:  two  cover-glass  method.     It  is  better 
to  place  the  top  cover  diagonally  and  to  grasp  it  by  opposite  covers. 

end  with  a  finger.     The  side  upon  which  it  twirls  the 
better  is  the  convex  side. 


Ehrlich'  sTwo  Cover-glass  Method. — This  method  is  very 
widely  used,  but  considerable  practice  is  required  to  get  good 
results.  Touch  a  cover-glass  to  the  top  of  a  small  drop  of 
blood,  and  place  it,  blood  side  down,  upon  another  cover- 
glass.  If  the  drop  be  not  too  large,  and  the  covers  be  per- 
fectly clean,  the  blood  wiU  spread  out  in  a  very  thin  layer. 
Just  as  it  stops  spreading,  before  it  begins  to  coagulate,  pull 
the  covers  quickly  but  firmly  apart  on  a  plane  parallel  to 
their  surfaces  (Fig.  112).'    It  is  best  to  handle  the  covers 


304 


THE  BLCOD 


with  forceps,  since  the  moisture  of  the  fingers  may  produce 
artifacts.     The  forceps  must  have  a  firm  grasp. 

This  method  is  especially  to  be  recommended  for  very 
accurate  differential  counts,  since  all  the  leukocytes  in 
the  drop  will  be  found  on  the  two  covers  and  thus  the 
possible  error  due  to  unequal  distribution  can  be  excluded. 
One  of  the  covers  is  usually  much  better  spread  than  the 
other. 

Two-slide  Method. — Take  a  small  drop  of  blood  upon  a 
clean  slide  about  ^  inch  from  the  end,  using  care  that  the 
slide  does  not  touch  the  skin.    Place  the  end  of  a  second 


Fig.  113. — Spreading  the  film:  two-slide  method. 


slide  against  the  surface  of  the  first  at  an  angle  of  30  to  40 
degrees,  and  draw  it  up  against  the  drop  of  blood,  which 
will  immediately  run  across  the  end,  filling  the  angle  be- 
tween the  two  slides.  Now  push  the  "spreader  slide" 
back  along  the  other  in  the  manner  indicated  in  Fig.  113. 
The  blood  will  follow.  The  thickness  of  the  smear  can  be 
regulated  by  changing  the  angle. 

It  is  very  easy  to  make  large,  thin,  even  films  by  this 
method. 

Cigarette-paper  Method.— This  gives  excellent  results  in 
the  hands  of  the  inexperienced  if  directions  are  carefully  fol- 
lowed, but  its  only  advantage  over  the  two-slide  method  is 


STUDY  OF  STAINED  BLOOD 


305 


that  it  may  be  used  with  covers  as  well  as  with  slides.  A 
very  thin  paper,  such  as  the  "Zig-zag"  brand,  is  best. 
Ordinary  cigarette  paper  and  thin  tissue-paper  will  answer, 
but  do  not  give  nearly  so  good  results. 

Cut  the  paper  into  strips  about  ^^  inch  wide,  across  the 
ribs.  Pick  up  one  of  the  strips  by  the  gummed  edge,  and 
touch  its  opposite  end  to  the  drop  of  blood.  Quickly  place 
the  end  which  has  the  blood  against  a  slide  or  a  large  cover- 


FiG.  114. 


-Spreading    the    film:  cigarette-paper    method    applied    to 
cover-glasses. 


glass  held  in  a  forceps.  The  blood  will  spread  along  the 
edge  of  the  paper.  Now  draw  the  paper  evenly  across 
the  slide  or  cover.  A  thin  film  of  blood  will  be  left  behind 
(Fig.  114). 

The  films  may  be  allowed  to  dry  in  the  air,  or  may  be 

dried  by  gently  warming  high  above  a  flame  (where  one 

can  comfortably  hold  the  hand).     Such  films  will  keep 

for  years,  but  for  some  stains  they  must  not  be  more 

than  a  few  weeks  old.     They  must  be  kept  away  from 

flies — a  fly  can  work  havoc  with  a  film  in  a  few  minutes. 
20 


306  THE   BLOOD 

When  slides  are  used  the  label  can  be  written  with  a 
soft  lead  pencil  directly  on  the  blood-film,  as  was  sug- 
gested by  von  Ezdorf. 

2.  Fixing  the  Film. — In  general,  films  must  be 
"fixed"  before  they  are  stained.  Fixation  may  be  ac- 
complished by  chemicals  or  by  heat.  Those  stains 
which  are  dissolved  in  methyl  alcohol  combine  fixation  with 
the  staining  process. 

Chemic  Fixation. — Soak  the  film  one  to  five  minutes  in 
pure  methyl  alcohol  or  absolute  ethyl  alcohol,  or  one-half 
hour  or  longer  in  equal  parts  of  absolute  alcohol  and  ether. 
One  minute  in  saturated  solution  of  mercuric  chlorid  or 


Fig.    115. — Kowarsky's    plate  for  fixing    blood   (Klopstock  and 
Kovvarsky) . 

in  I  per  cent,  formalin  in  alcohol  is  preferred  by  some, 
especially  for  the  carbol-thionin  stain.  Chemic  fixation 
may  precede  hematoxylin-eosin  and  other  simple  stains. 

Heat  Fixation.— This  may  precede  any  of  the  methods 
which  do  not  combine  fixation  with  the  staining  process;  it 
is  almost  imperative  with  Ehrlich's  triple  stain.  The  best 
method  is  to  place  the  film  in  an  oven,  raise  the  temperature 
to  i5o°C.,  and  allow  to  cool  slowly.  Without  an  oven, 
the  proper  degree  of  fixation  is  difficult  to  attain.  Kow- 
arsky  has  devised  a  small  plate  of  two  layers  of  copper 
(Fig.  115),  upon  which  the  films  are  placed  together  with  a 
crystal  of  urea.  The  plate  is  heated  over  a  flame  until  the 
urea  melts,  and  is  then  set  aside  to  cool.     Some  prefer,  to 


STUDY    OF    STAINED  BLOOD  307 

use  slides  and  to  place  the  crystal  of  urea  directly  upon  the 
slide.  This  is  said  to  give  the  proper  degree  of  fixation, 
but  in  the  writer's  experience  the  films  have  always  been 
underheated.  He  obtains  better  results  by  use  of  tar- 
taric acid  crystals  (melting-point,  i68°-i7o°C.).  The 
plate,  upon  which  have  been  placed  the  cover-glasses,  film 
side  down,  and  a  crystal  of  the  acid,  is  heated  over  a  low 
flame  until  the  crystal  has  completely  melted.  It  should 
be  held  sufl&ciently  high  above  the  flame  that  the  heating 
will  require  five  to  seven  minutes.  The  covers  are  then 
removed.  Freshly  made  films  of  normal  blood  should  be 
allowed  to  remain  upon  the  plate  for  a  minute  or  two  after 
heating  has  ceased.  Fresh  films  require  more  heat  than  old 
ones,  and  normal  blood  .more  than  the  blood  of  pernicious 
anemia  and  leukemia. 

Blood-films  can  be  satisfactorily  fixed  for  most  purposes 
by  covering  with  absolute  alcohol,  quickly  dashing  off  the 
excess,  and  igniting  the  remainder. 

3.  Staining  the  Film. — The  anilin  dyes,  which  are 
extensively  used  in  blood  work,  are  of  two  general 
classes:  basic  dyes,  of  which  methylene-blue  is  the  type; 
and  acid  dyes,  of  which  eosin  is  the  type.  Nuclei  and 
certain  other  structures  in  the  blood  are  stained  by  the 
basic  dyes,  and  are  hence  called  basophilic.  Certain 
structures  take  up  only  acid  dyes,  and  are  called  acido- 
philic, oxyphilic,  or  eosinophilic.  Certain  other  struc- 
tures are  stained  by  combinations  of  the  two,  and 
are  called  neutrophilic.  Recognition  of  these  staining . 
properties  marked  the  beginning  of  modern  hematology. 

(i)  Hematoxylin  and  Eosin. — This  method  is  most 
useful  in  studying  eosinophilic  cells  and  the  structure  of 
nuclei,  hematoxylin  being  in  fact  one  of  our  best  nu- 
clear stains.     It  may  therefore  be  recommended  for  the 


3o8  THE  BLOOD 

Arneth  count  (see  p.  334).  Red  corpuscles  are  pink  or 
red,  all  nuclei  blue,  eosinophilic  granules  bright  red; 
neutrophilic  granules  and  platelets  are  not  stained. 
Neither  polychromatophilia  nor  basophilic  granular 
degeneration  of  the  red  cells  are  demonstrated. 

1.  Fix  by  heat  or  chemicals. 

2.  Stain  with  any  standard  hematoxylin  solution  until 
nuclei  are  well  colored,  usually  three  to  five  minutes. 

3.  Wash  well  with  water. 

4.  Apply  a  weak  aqueous  or  alcoholic  solution  of  eosin 
(about  0.5  per  cent.)  for  a  minute  or  two. 

5.  Wash  well  in  water,  dry  and  examine.  If  the  eosin 
stains  too  deeply,  longer  washing  in  water  will  usually  re- 
move some  of  the  excess. 

The  procedure  may  be  simplified  by  mixing  the  hema- 
toxylin and  eosin.  Such  a  mixture  was  much  used  before 
modern  staining  methods  were  introduced.  Almost  any 
of  the  standard  hematoxylin  solutions  may  be  employed; 
to  this  is  added  enough  of  a  saturated  aqueous  solution  of 
eosin  to  color  the  reds  properly  while  the  hematoxylin  is 
staining  the  nuclei.  The  combined  stain  keeps  well.  The 
fixed  smear  is  immersed  in  the  staining  fluid  for  the  re- 
quired time,  usually  five  to  fifteen  minutes,  and  is  then 
rinsed,  dried,  and  mounted. 

(2)  Ehrlich's  Triple  Stain.- — This  was  the  standard 
blood-stain  for  many  years,  but  is  now  little  used.  It 
is  probably  the  best  for  neutrophilic  granules.  It  is 
difficult  to  make,  and  should  be  purchased  ready  pre- 
pared from  a  reliable  dealer.  Nuclei  are  stained  pale 
blue  or  greenish  blue;  erythrocytes,  orange;  neutro- 
philic granules,  violet;  and  eosinophilic  granules,  copper 
red.  Basophilic  granules  and  blood-platelets  are  not 
stained. 


STUDY  OF  STAINED  BLOOD  309 

Success  in  staining  depends  largely  upon  proper  fixa- 
tion. The  film  must  be  carefully  fixed  by  heat :  under- 
heating  causes  the  erythrocytes  to  stain  red;  overheat- 
ing, pale  yellow.  Immersion  in  pure  acetone  for 
five  minutes  has  been  recommended  as  a  satisfactory 
substitute  for  heat  fixation.  The  staining  fluid  is 
applied  for  five  to  fifteen  minutes,  and  the  preparation 
is  rinsed  quickly  in  water,  dried,  and  mounted.  Sub- 
sequent application  of  LoiHer's  methylene-blue  for 
one-half  to  one  second  will  bring  out  the  basophilic 
granules  and  improve  the  nuclear  staining,  but  there 
is  considerable  danger  of  overstaining.  The  fluid 
should  not  be  filtered  regardless  of  any  precipitate  that 
may  form. 

(3)  Polychrome  Methylene-blue -eosin  Stains.^ — 
These  stains,  outgrowths  of  the  original  time-consum- 
ing Romanowsky  method,  have  largely  displaced  other 
blood-stains  for  clinical  purposes.  They  may  be  recom- 
mended for  all  routine  work.  They  stain  differentially 
every  normal  and  abnormal  structure  in  the  blood. 
Most  of  them  are  dissolved  in  methyl  alcohol  and  com- 
bine the  fixing  with  the  staining  process.  Numer- 
ous methods  of  preparing  and  applying  these  stains 
have  been  devised,  among  the  best  known  being 
Giemsa's,  Wright's,  Hastings'  and  Leishman's. 

Wright's  Stain.^ — This  is  one  of  the  best  and  is  the 
most  widely  used  in  this  country.  The  practitioner 
will  find  it  convenient  to  purchase  the  stain  ready  pre- 
pared, but,  since  much  of  the  solution  offered  for  sale 
is  unsatisfactory,  it  is  best  to  purchase  the  powder  and 
dissolve  it  in  methyl  alcohol  as  needed.  Most  micro- 
scopic supply-houses  carry  it  in  stock.     Wright's  most 


3IO  THE   BLOOD 

recent  directions  for  its   preparation   and  use   are  as 
follows : 

Preparation. — To  a  0.5  per  cent,  aqueous  solution  of 
sodium  bicarbonate  add  methylene-blue  (B.  X.  or  "medicin- 
ally pure")  in  the  proportion  of  i  Gm.  of  the  dye  to  each  100 
c.c.  of  the  solution.  Heat  the  mixture  in  a  steam  sterilizer 
at  ioo°C.  for  one  full  hour,  counting  the  time  after  the  ster- 
ilizer has  become  thoroughly  heated.  The  mixture  is  to  be 
contained  in  a  flask,  or  flasks,  of  such  size  and  shape  that  it 
forms  a  layer  not  more  than  6  cm.  deep.  After  heating, 
allow  the  mixture  to  cool,  placing  the  flask  in  cold  water, 
if  desired,  and  then  filter  it  to  remove  the  precipitate  which 
has  formed  in  it.  It  should,  when  cold,  have  a  deep  purple- 
red  color  when  viewed  in  a  thin  layer  by  transmitted  yellow- 
ish artificial  light.  It  does  not  show  this  color  while  it  is 
warm. 

To  each  100  c.c.  of  the  filtered  mixture  add  500  c.c.  of  a 
0.1  per  cent,  aqueous  solution  of  "yellowish  water-soluble" 
eosin  and  mix  thoroughly.  Collect  the  abundant  precipi- 
tate, which  immediately  appears,  on  a  filter.  When  the  pre- 
cipitate is  dry,  dissolve  it  in  methylic  alcohol  (Merck's 
"reagent")  in  the  proportion  of  o.i  Gm.  to  60  c.c.  of  the 
alcohol.  In  order  to  facilitate  solution,  the  precipitate  is 
to  be  rubbed  up  with  the  alcohol  in  a  porcelain  dish  or 
mortar  with  a  spatula  or  pestle.  This  alcoholic  solution 
of  the  precipitate  is  the  staining  fluid.  We  frequently  find 
that  freshly  made  solutions  stain  the  red  cells  blue;  such 
solutions  usually  work  properly  after  a  few  months. 

Application. — i.  Without  previous  fixation  cover  the  film 
with  a  noted  quantity  of  the  staining  fluid  by  means  of  a 
medicine-dropper.  There  must  be  plenty  of  stain  in  order 
to  avoid  too  great  evaporation  and  consequent  precipita- 
tion. When  slides  are  used,  the  stain  may  be  confined  to 
the  smeared  area  by  two  heav>'  wax  pencil  marks. 


STUDY    OF    STAINED  BLOOD  3II 

2.  After  one  minute  add  to  the  staining  fluid  on  the  film 
the  same  quantity  of  distilled  water  by  means  of  a  medicine- 
dropper.  This  may  be  done  by  counting  drops.  The 
drops  of  water  are  about  twice  as  large  as  the  drops  of 
stain,  but  this  rarely  does  any  harm  and  is  often  an  ad- 
vantage. The  quantity  of  the  fluid  on  the  preparation 
must  not  be  so  large  that  some  of  it  runs  off.  Allow  the 
mixture  to  remain  for  three  to  six  minutes,  according  to 
the  intensity  of  the  staining  desired.  A  longer  period  of 
staining  may  produce  a  precipitate.  Eosinophilic  granules 
are  best  brought  out  by  a  short  period  of  staining. 

3.  Wash  the  preparation  in  water  for  thirty  seconds  or 
until  the  thinner  portions  of  the  film  become  yellow  or 
pink  in  color.  .  The  preparation  should  be  flooded  with 
water  while  the  stain  is  still  upon  it.  If  the  stain  is  poured 
off  before  rinsing,  the  scum  tends  to  settle  upon  the  blood- 
film,  where  it  clings  in  spite  of  subsequent  washing. 

4.  Dry  and  mount  in  balsam. 

The  stain  is  more  conveniently  applied  upon  cover- 
glasses  than  upon  slides.  Films  much  more  than  a 
month  old  do  not  stain  well,  red  cells  and  most  other 
structures  taking  a  slate  blue  color.  .  In  some  localities 
ordinary  tap-water  will  answer  both  for  diluting  the 
stain  and  for  washing  the  film;  in  others,  distilled 
water  must  be  used.  The  difficulty  here  is  probably 
that  the  tap-water  is  acid  in  reaction.  This  causes 
the  nuclei  to  stain  too  palely.  Other  causes  of  pale 
nuclei  are  addition  of  too  much  or  too  little  water 
and  the  development  of  formic  acid  from  the  methyl 
alcohol  of  the  staining  fluid. 

When  properly  applied,  Wright's  stain  gives  the  fol- 
lowing picture  (see  Plates  I,  V,  VII):  erythrocytes, 
yellow  or  pink;  nuclei,  various  shades  of  bluish  purple; 


312  THE   BLOOD 

neutrophilic  granules,  reddish  lilac,  sometimes  pink; 
eosinophilic  granules,  bright  red;  basophilic  granules  of 
leukocytes  and  degenerated  red  corpuscles,  very  dark 
bluish  purple;  blood-platelets,  dark  lilac;  bacteria,  blue. 
The  cytoplasm  of  lymphocytes  is  generally  robin's-egg 
blue;  that  of  the  large  mononuclears  may  have  a  faint 
bluish  tinge.  Malarial  parasites  stain  characteristically : 
the  cytoplasm,  sky-blue;  the  chromatin,  reddish  purple. 
These  colors  are  not  invariable:  two  films  stained 
from  the  same  bottle  sometimes  differ  greatly.  In 
general  a  preparation  is  satisfactory  when  both  nticlei 
and  neutrophilic  granules  are  distinct,  regardless  of 
their  color,  and  when  the  film  is  free  from  precipitated  dye. 
In  addition,  it  is  desirable,  but  not  essential,  that  the 
red  corpuscles  show  a  clear  pink  or  yellowish-pink; 
they  should  not  be  blue.  The  colors  are  prone  to  fade  if 
the  preparation  is  mounted  in  a  poor  quality  of  balsam 
or  exposed  much  to  the  light. 

It  is  well  known  that  pathologic  bloods  will  sometimes 
not  stain  well  with  fluids  which  are  satisfactory  for 
normal  blood.  Peebles  and  Harlow  have  shown  that 
the  various  polychrome  methylene-blue-eosin,  stains 
can  be  modified  to  suit  any  blood  by  adding  a  trace  of 
alkali  or  acid.  The  alkali  used  is  a  weak  solution  of 
''potassium  hydrate  by  alcohol"  in  methyl  alcohol; 
the  acid,  glacial  acetic  in  methyl  alcohol.  The  alkali 
solution  also  serves  to  "correct"  old  fluids  which,  by 
reason  of  development  of  formic  acid  in  the  methyl 
alcohol,  do  not  stain  sufficiently  with  the  blue. 

Other  Polychrome  Methylene -blue-eosin  Stains. — While 
Wright's  stain  suffices  for  most  clinical  work  and  is  to  be 


STUDY    OF   STAINED  BLOOD  313 

recommended  if  only  one  blood  stain  is  to  be  used,  certain 
others  demand  brief  mention. 

1.  Giemsa's  Stain. — This  widely  used  stain  is  prob- 
ably the  best  modification  of  the  Romanowsky  stain  for  blood 
parasites  and  other  protozoa,  and  is  also  very  satisfactory 
as  a  routine  blood  stain.     It  consists  of: 

Azur  Il-eosin 3  Gm. 

Azur   II 0.8  Gm. 

Glycerin  (Merck,  C.  P.) 250  Gm. 

Methyl    alcohol     (Kahlbaum     I     or 

Merck's  reagent) 250  Gm. 

The  solution  is  expensive  to  make  and  is  best  purchased 
ready  prepared.  Blood  films  are  fixed  in  methyl  alcohol 
and  are  then  immersed  for  twenty  minutes  or  longer  in  a 
freshly  prepared  mixture  of  i  c.c.  of  stain  and  10  c.c. 
distilled  water.  In  order  to  prevent  precipitates  falling 
upon  them,  the  slides  or  covers  should  be  placed  upon  edge 
in  the  stain. 

The  use  of  this  stain  for  Treponema  pallidum  is  described 
later  (p.  550). 

2.  Pappenheim's  Panoptic  Method. — In  order  to  com- 
bine the  advantages  of  the  several  stains,  Pappenheim 
recommended  the  following  procedure:  Stain  for  one  min- 
ute with  the  May-Griinwald  stain;  add  an  equal  quantity 
of  water;  after  one  minute  pour  off  the  fluid  and  stain 
fifteen  minutes  with  the  diluted  Giemsa  solution.  The 
May-Griinwald  stain  is  the  same  as  Jenner's.  Wright's 
stain,  diluted  with  an  equal  quantity  of  water,  may  be 
substituted  for  the  Giemsa  solution  but  the  time  of  staining 
should  then  not  exceed  five  mmutes. 

It  is  difficult  to  see  that  slides  stained  in  this  way  ofifer 
any  advantages  over  good  Wright  or  Giemsa  preparations. 

(4)  Jenner's  Stain. — Jenner's  eosinate  of  methylene- 
blue,  dissolved  in  methyl  alcohol,  brings  out  leukocytic 


314  THE   BLOOD 

granules  well,  and  is,  therefore,  especially  useful  for 
differential  counting.  It  stains  nuclei  poorly  and  is 
much  inferior  to  Wright's  stain  for  the  malarial  parasite 
since  it  does  not  give  the  so-called  " Romano wsky 
staining." 

It  may  be  purchased  in  solution,  in  the  form  of  tablets, 
or  as  a  powder,  0.5  Gm.  of  which  is  to  be  dissolved  in 
100  c.c.  neutral  absolute  methyl  alcohol.  The  unfixed 
blood-film  is  covered  with  the  staining  solution  and 
after  three  to  five  minutes  is  rinsed  with  water,  dried 
in  the  air,  and  mounted. 

(5)  Carbol-thionin  is  especially  useful  for  the  study 
of  basophilic  granular  degeneration  of  the  red  cells. 
The  method  is  described  on  page  639.  Nuclei,  malarial 
parasites,  and  basophilic  granules  are  brought  out 
sharply.  Polychromatophilia  is  also  evident.  Fixa- 
tion may  be  by  alcohol-formaUn  (see  p.  306)  or  satu- 
rated solution  of  mercuric  chlorid. 

(6)  Pappenheim's  pyronin-methyl  green  (see  p.  642) 
can  be  used  as  a  blood-stain  and  is  very  satisfactory  for 
study  of  the  red  cells  and  of  the  lymphocytes  and  for 
demonstration  of  Doehle's  inclusion  bodies  (see  p.  335). 
All  nuclei  are  blue  to  reddish  purple;  basophilic 
granules,  cytoplasm  of  lymphocytes,  and  inclusion 
bodies,  red.  Polychromatophilia  is  well  demonstrated, 
the  affected  cells  taking  more  or  less  of  the  red  color. 
Heat  fixation  is  probably  best. 

B.  Study  of  Stained  Films 

It  has  been  said  with  much  truth  that  an  intelligent 
study  of  the  stained  film,  together  with  an  estimation  of 
hemoglobin,  will  yield  90  per  cent,  of  all  the  diagnostic 


STUDY   OF    STAINED  BLOOD  315 

information  obtainable  from  a  blood-examination. 
The  stained  films  furnish  the  best  means  of  studying 
the  morphology  of  the  blood  and  blood  parasites,  and, 
to  the  experienced,  they  give  a  fair  idea  of  the  amount 
of  hemoglobin  and  the  number  of  red  and  white  cor- 
puscles.    An  oil-immersion  objective  is  required. 


Fig.   116. — Red  corpuscles  of  normal  blood.      Wright's  stain  (X  7So). 

1.  Erythrocytes. — Normally,  the  red  corpuscles  are 
acidophilic.  The  colors  which  they  take  with  different 
stains  have  been  described.  When  not  crowded  to- 
gether, they  appear  as  circular,  homogeneous  disks,  of 
nearly  uniform  size,  averaging  7.8  /i  in  diameter  (see 
Fig.  116),  The  center  of  each  is  somewhat  paler  than 
the  periphery.  Red  cells  are  apt  to  be  crenated  when 
the  film  has  dried  too  slowly. 


3l6  THE  BLOOD 

Pathologically,  red  corpuscles  vary  inherrroglobin  con- 
tent, size  and  shape,  staining  properties,  and  structure, 
(i)  Hemoglobin  Content — The  depth  of  staining 
furnishes  a  rough  guide  to  the  amount  of  hemoglobin  in 
the  corpuscles,  i.e.,  to  the  color  index.  WTien  hemo- 
globin is  diminished  the  central  pale  area  becomes 
larger  and  paler.  This  condition  is  known  as  a^hromiu. 
Usually  the  periphery  retains  a  fairly  deep  color,  so 
that  the  cells  become  mere  rings,  the  so-called  ''pessary- 
forms.''     These   are   most   common  in   chlorosis.     In 


Fig.  117. — Red    blood-corpuscles    showing    deficient    hemoglobin 
(achromia).     From   a  "wrell  marked  case  of  chlorosis.     Wright's  stain 

(X  750). 


pernicious  anemia,  upon  the  other  hand,  as  a  result  of 
the  high  color  index  many  of  the  red  corpuscles  may 
stain  deeply  and  lack  the  pale  center  entirely. 

(2)  Variations  in  Size  and  Shape  (See  Plate  V,  Fig.  i) . 
— The  cells,  may  be  abnormally  small  (called  mkrocytes^ 
S  fi  OT  less  in  diameter) ;  abnormally  large  {macrocytes, 
10  to  12  /i) ;  or  extremely  large  {megalccytes,  12  to  25  /*). 
Abnormal  variation  in  size  is  called  anisocytosis. 

Variation  in  shape  is  often  very  marked.  Oval,  pyri- 
form,  caudate,  saddle-shaped,  and  club-shaped  corpus- 


Description  of  Plate  V 

Abnormal  red  corpuscles.     All  drawn  from  actual  specimens  and  all  stained  with  Wright's 

stain  except  where  noted,      x  looo  (i  mm.  =  i  micron). 

Fig.  I. — Variations  in  size,  shape,  and  hemoglobin-content;  from  cases  of  pernicious 
anemia  and  chlorosis. 

Fig.  2. — Polychromatophilia  and  basophilic  granular  degeneration;  from  cases  of  lead- 
poisoning  and  pernicious  anemia. 

Fig.  3. — Normoblasts,  reticulated  red  cells,  and  one  microblast.  The  top  row  repre- 
sents stages  in  the  development  of  the  normoblast.  The  two  reticulated  red  cells  arc 
staineil  with  brilliant  cresyl  blue. 

Fig.  4. — Mcgaloblasts  from  cases  of  pernicious  anemia.  Two  show  p<ilychromato- 
philia  and  fairly  typical  nuclei,  two  have  condensed  nuclei,  and  one  of  these  has  basophilic 
cytoplasmic  granules. 

Fig.  5. — Nuclear  particles  or  "Howell-Jolly  bodies."  One  cell  ako  shows  basophilic 
granular  degeneration. 

Fig.  6. — l^itotic  figures,  two  from  myelogenous  leukemia,  one,  with  jxjlychromato- 
pliilic  Cytoplasm,  from  von  Jaksch's  anemia.    The  lust  was  stained  with  Leishman's  stain. 

Fig.  7. — Cabot's  ring  bodies,  from  a  case  of  von  Jaksch's  anemia.  Two  cells  also  con- 
tain nuclear  particles  and  one  shows  basophilic  granular  defeneration.     Leishman's  stain. 


PLATE  V 


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STUDY  OF  STAINED  BLOOD  317 

cles  are  common  (Fig.  ii8).  They  are  called  poikilo- 
cytes,  and  their  presence  is  spoken  of  as  poikilocytosis. 
Red  corpuscles  which  vary  from  the  normal  in  size 
and  shape  are  present  in  most  symptomatic  anemias, 
and  in  the  severer  grades  are  often  very  numerous. 
Irregularities  are  particularly  conspicuous  in  leukemia 
and  pernicious  anemia,  where,  in  some  instances,  a  nor- 
mal erythrocyte  is  the  exception.  In  pernicious  anemia 
there  is  a  decided  tendency  to  large  size  and  oval  forms, 
and  megalocytes  are  rarely  found  in  any  other  condition. 


^?f-:.zA  ^srsssrs; 


^ 


^,/   «  '%^.#. 

^'iG.   118. — Red  corpuscles  showing  variations  in.  size  and  shape,  from 
a  case  of  pernicious  anemia  (  X  750). 

(3)  Variations  in  Staining  Properties  (See  Pkte  V, 
Fig.  2). — These  include  polychromatophilia,  basophilic 
granular  degeneration,  and  malarial  stippling.  With 
exception  of  polychromatophilia  they  are  probably 
degenerative  changes. 

(a)  Polychromatophilia. — Some  of  the  corpuscles  par- 
tially lose  their  normal  affinity  for  acid  stains  and  take 
the  basic  stain  to  greater  or  less  degree.  Wright's  stain 
gives  such  cells  a  faint  bluish  tinge  when  the  condition  is 
mild,  and  a  rather  deep  blue  when  severe.     Sometimes 


3l8  THE  BLOOD 

only  part  of  a  cell  is  affected.  A  few  polychromato- 
philic  corpuscles  can  be  found  in  marked  symptomatic 
anemias.  They  occur  most  abundantly  in  malaria, 
leukemia,  and  pernicious  anemia. 

Polychromatophilia  has  been  variously  interpreted. 
It  is  thought  by  many  to  be  evidence  of  youth  in  a  cell, 
and  hence  to  indicate  an  attempt  at  blood  regeneration. 
There  are  probably  several  forms  referable  to  different 
causes. 

{b)  Basophilic  Granular  Degeneration  {Degeneratio^i  oj 
Graivitz,  Basophilic  Stippling). — This  is  characterized 


i 


€tB 


Fig.   119. — Red  blood-corpuscle  showing   basophilic  granular  degen- 
eration with  large  granules.      Wright's  stain  (X  1000). 

by  the  presence,  within  the  corpuscle,  of  irregular  baso- 
philic granules  which  vary  in  size  from  scarcely  visible 
points  to  granules  nearly  as  large  as  those  of  basophilic 
leukocytes  (Fig.  119).  The  number  present  in  a  red  cell 
commonly  varies  in  inverse  ratio  to  their  size.  They 
stain  deep  blue  with  carbol-thionin  or  Wright's  stain; 
not  at  all  with  Ehrlich's  triple  stain.  The  cell  con- 
taining them  may  stain  normally  in  other  respects, 
or  it  may  exhibit  polychromatophilia.  Polychromato- 
philic  cells  generally  contain  the  smaller  granules,  which 
may  be  so  fine  that  the  cell  appears  dusted  with  them. 


STUDY  OF  STAINED  BLOOD 


319 


Numerous  cells  showing  this  degeneration  are  com- 
monly found  in  chronic  lead-poisoning,  of  which  they 
were  at  one  time  thought  to  be  pathognomonic.  They 
can  probably  be  found  in  every  case  with  clinical  symp- 
toms and  in  some  severe  cases  are  present  in  nearly 
every  microscopic  field.  Except  in  this  disease,  the 
degeneration  indicates  a  serious  blood  condition.  It 
occurs  in  well-marked  cases  of  pernicious  anemia  and 
leukemia,  and,  much  less  commonly,  in  very  severe 
symptomatic  anemias. 


Fig.   120. — Normoblasts  from  cases  of  secondary  anemia  and  leukemia 
(  X  1000). 


(c)  Malarial  Stippling. — This  term  has  been  applied 
to  the  finely  granular  appearance  often  seen  in  red  cor- 
puscles which  harbor  tertian  malarial  parasites  (see 
Frontispiece,  Plates  VI  and  VII).  It  was  formerly 
classed  with  the  degeneration  just  described,  but  is 
undoubtedly  distinct.  Not  all  stains  will  show  it. 
With  Wright's  stain  it  can  be  brought  out  by  staining 
longer  and  washing  less  than  for  the  ordinary  blood- 
stain. The  minute  granules,  "Schufi"ner's  granules," 
stain  reddish  purple.  They  are  sometimes  so  numerous 
as  almost  to  hide  the  parasite. 


320  THE  BLOOD 

(4)  Variations  in  Structure.^The  most  important  Is 
the  presence  of  a  nucleus  (see  Frontispiece,  Plate  V, 
and  Figs.  3-7).  Nucleated  red  corpuscles,  or  erythro- 
blasts,  are  classed  according  to  their  size:  microhlasts, 
5  /*  or  less  in  diameter;  normoblasts,  5  to  10  /*;  and 
megaloblasts,  above  10  /x. 

Microblasts  and  normoblasts  contain  one,  rarely 
two,  small,  round,  sharply  defined  nuclei.  As  a  rule 
they  are  the  most  deeply  stained  nuclei  to  be  seen  in 
the  blood-film,  being  approached  in  this  respect  only  by 


Fig.   121. — Normoblasts     with     irregular     and      fragmented     nuclei. 
Wright's  stain  (  X  looo). 

the  smaller  lymphocytes.  The  nuclei  of  the  younger 
normoblasts  are  relatively  large  and  have  their  chro- 
matin arranged  in  a  more  or  less  reticular  manner  with 
rather  clean-cut  open  spaces.  Mitoses  are  not  uncom- 
mon in  leukemia  and  pernicious  anemia.  The  older 
nuclei  are  smaller  and  more  dense,  some  being  entirely 
homogeneous  and  very  deeply  stained  (pyknotic  nuclei) . 
These  last  are  apt  to  be  located  eccentrically,  and 
sometimes  appear  as  if  in  process  of  extrusion  from 
the  cell.  These  characteristics  are  shown  in  Fig.  120. 
As  a  result  of  degenerative  changes  the  nuclei  may  be 


STUDY    OF    STAINED   BLOOD  32 1 

irregular  in  shape,  clover-leaf  forms  being  common;  or 
they  may  be  completely  broken  up  into  fragments— 
the  so-called  nuclear  particles  or  Howell- Jolly  bodies 
— of  which  all  but  one  or  two  may  have  disappeared 
from  the  cell.  These  nuclear  particles  are  smooth, 
round,  deeply  stained  bodies,  not  likely  to  be  mis- 
taken for  granules  of  basophilic  degeneration  (Fig.  122). 
The  megaloblast  is  probably  a  distinct  cell,  not 
merely  a  larger  size  of  the  normoblast.     In  the  typical 


Fig.   122. —  Nuclear  particles  or  Howell-JoUy  bodies  in  red  corpuscles. 
From  a  case  of  pernicious  anemia.     Wright's  stain  (  X   looo). 

megaloblast  the  nucleus  is  characteristic.  This  is  large, 
oval,  and  rather  palely  staining  and  it  has  a  more  delicate 
chromatin  network  with  larger  and  more  numerous 
openings  than  has  the  nucleus  of  the  normoblast  (see 
Plates  V,  VIII,  and  Fig.  123).  Sometimes  it  appears 
as  if  made  up  of  coarse  granules.  Evidences  of  age 
and  degeneration  (condensation  of  nucleus,  pyknosis, 
karyorrhexis,  etc.),  are  common. 

The  recognition  of  megaloblasts  is  important,  but  is 
not  always  easy  unless  the  nucleus  is  typical.  Some 
workers  base  the  distinction  from  normoblasts  upon  size 
of  nucleus,  requiring  this  to  be  larger  than  a  normal  red 


322 


THE   BLOOD 


corpuscle  if  the  cell  is  to  be  regarded  as  a  megaloblast. 
Others  consider  only  the  size  of  the  cell,  regarding  as  a 
megaloblast  any  nucleated  red  cell  over  ii  /^  in  diameter. 
Neither  of  these  rules,  nor  the  two  together,  will  serve 
in  every  case.  The  exceptions  will  include,  on  the 
one  hand,  certain  old  megaloblasts  with  small  condensed 
nuclei,  and  upon  the  other,  the  very  young  normo- 
blast whose  diameter  may  exceed  1 2  /i  and  whose  nuclei 
may  be  larger  than  a  normal  red  corpuscle.  At  times 
one  finds  cells  which  must  be  classed  as  intermediates. 


Fig.   123.- — Megaloblasts  showing  typical  nuclei;  from  cases  of  perni- 
cious anemia.     Wright's  stain  (  X  looo). 

Young  nucleated  red  cells,  especially  megaloblasts 
are  prone  to  exhibit  polychromatophilia.  In  some 
cells  the  cytoplasm  is  so  blue  and  shows  so  little 
of  its  characteristic  smooth  texture  that  it  is  diffi- 
cult to  recognize  the  cell  as  an  erythrocyte  except  by 
the  character  of  the  nucleus.  Such  cells  might  easily 
be  niistaken  for  lymphocytes  or  for  Tiirck's  irritation 
leukocytes. 

Significance  of  Nucleated  Red  Corpuscles. — 
Normally,  erythroblasts  are  present  only  in  the  blood 


STUDY  OF  STAINED  BLOOD  323 

of  the  fetus  and  of  very  young  infants.  In  the  healthy 
adult  they  are  confined  to  the  bone-marrow  and  they 
appear  in  the  circulating  blood  only  in  disease,  where 
their  presence  denotes  an  excessive  demand  made 
upon  the  blood-forming  organs  to  regenerate  lost 
or  destroyed  red  corpuscles.  In  response  to  this 
demand  immature  and  imperfectly  formed  cells  are 
thrown  into  the  circulation.  Their  number,  therefore, 
is  usually  regarded  as  an  indication  of  the  extent  to 
which  the  bone-marrow  reacts  rather  than  of  the  sever- 
ity of  the  disease.  Normoblasts  occur  in  severe  symp- 
tomatic anemia,  leukemia,  and  pernicious  anemia. 
They  are  most  abundant  in  myelogenous  leukemia. 
While  always  present  in  pernicious  anemia,  they  are 
often  difficult  to  find.  Microblasts  have  much  the  same 
significance  as  normoblasts,  but  are  less  common. 
Nuclear  particles,  or  Howell- Jolly  bodies,  are  most  com- 
mon in  pernicious  anemia  and  have  been  noted  in 
greatest  numbers  after  splenectomy. 

The  presence  of  megaloblasts  indicates  a  change  in 
the  type  of  blood  regeneration.  This  is  seen  most 
characteristically  in  pernicious  anemia  and  the  finding 
of  megaloblasts  is  therefore  extremely  important  in  the 
diagnosis  of  this  disease.  They  are  always  present, 
although  often  in  so  small  numbers  as  to  require  a  long 
search;  and  they  almost  invariably  exceed  the  normo- 
blasts in  number — a  ratio  which  is  practically  unknown 
in  other  diseases  in  which  they  have  been  found,  such 
as  myelogenous  leukemia,  malignant  growths  in  the 
bone-marrow,  etc. 

Cabot's  ring  bodies  are  ring-  or  figure-of-8-shaped 
structures  (Fig.  124)  which  have  been  observed  in  cer- 


324  THE   BLOOD 

tain  of  the  red  cells  in  pernicious  anemia,  lead-poisoning, 
and  lymphatic  leukemia.  They  stain  red  or  reddish 
purple  with  Wright's  stain  and  have  been  thought  to 
be  the  remains  of  a  nuclear  membrane. 

2.  The  Leukocytes. — An  estimation  of  the  number 
or  percentage  of  each  variety  of  leukocyte  in  the  blood 
is  called  a  differential  count.  It  probably  yields  more 
helpful  information  than  any  other  single  procedure  in 
blood  examinations. 

The  differential  count  is  best  made  upon  a  film 
stained   with   Jenner's,    Wright's,   or    a  similar  stain. 


Fig.  124. — Cabot's  ring  bodies  in  red  blood-corpuscles  from  a  case  of 
von  Jaksch's  anemia  of  infancy.  The  cell  at  the  right  contains  a  ring, 
a  nuclear  particle,  and  basophilic  granules.     Leishman's  stain  (  X  looo). 


Wright's  stain  is  probably  most  widely  used  but  dif- 
ferentiates the  leukocytes  somewhat  less  satisfactorily 
than  Jenner's  or  Ehrlich's.  The  blood-film  need  not 
be  quite  so  thin  as  is  required  for  study  of  the  red  cells, 
but  it  must  be  thin  enough  to  enable  one  to  identify 
the  leukocytes  without  difficulty.  One  should  first 
glance  over  the  preparation  to  find  what  the  general 
tinting  of  the  cells  may  be.  Two  films  stained  side  by 
side  will  often  show  marked  differences  in  the  color  re- 
actions of  the  cells. 


STUDY  OF  STAINED  BLOOD  325 

To  make  the  differential  count  go  carefully  over  the 
film  with  an  oil-immersion  lens,  using  a  mechanical 
stage  if  available.  Experienced  workers  often  use  the 
lower  powers  (even  the  i6-mm.,  as  recommended  by 
Simon)  in  routine  work;  but  the  film  must  then  be 
mounted,  or  wet  with  water  or  oil  since  these  lenses 
cannot  be  used  satisfactorily  upon  dry,  unmounted  films. 
Classify  each  leukocyte  seen,  and  calculate  what  per- 
centage each  variety  is  of  the  whole  number  classified. 
For  accuracy,  500  to  1000  leukocytes  must  be  classified; 
for  approximate  results,  300  are  sufficient,  but  it  is  im- 
perative to  count  cells  in  all  parts  of  the  smear,  since  the 
different  varieties  of  leukocytes  may  be  unevenly  dis- 
tributed. Track  of  the  count  may  be  kept  by  placing 
a  mark  for  each  leukocyte  in  its  appropriate  column, 
ruled  upon  paper.  Some  workers  divide  a  slide-box 
into  compartments  with  slides,  one  for  each  variety  of 
leukocyte,  and  drop  a  coffee-bean  into  the  appropriate 
compartment  when  a  cell  is  classified.  When  a  con- 
venient number  of  coffee-beans  is  used  (any  multiple  of 
100),  the  percentage  calculation  is  simple. 

The  actual  number  of  each  variety  in  a  cubic  milli- 
meter of  blood  is  easily  calculated  from  these  percent- 
ages and  the  total  leukocyte  count,  and  should  form 
part  of  the  record  if  this  is  to  be  complete.  An  increase 
in  actual  number  is  an  absolute  increase;  an  increase  in 
percentage  only,  a  relative  increase.  It  is  evident  that 
an  absolute  increase  of  any  variety  may  be  accompanied 
by  a  relative  decrease. 

One  should  make  it  a  rule,  when  making  a  differential 
count,  always  to  attempt  to  estimate  the  total  leuko- 
cyte count  from  the  appearance  of  the  stained  film 


326  THE  BLOOD 

with  the  low-power  objective.  After  some  practice, 
this  can  be  done  with  a  considerable  degree  of  accuracy. 

The  number  of  nucleated  red  corpuscles  seen  while 
making  the  count  is  generally  included  in  the  record. 

The  usual  classification  of  leukocytes  is  based  upon 
their  size,  their  nuclei,  and  the  staining  properties  of 
the  granules  which  many  of  them  contain.  It  is  not 
altogether  satisfactory,  but  is  probably  the  best  which 
our  present  knowledge  permits.  The  leukocytes  of 
normal  blood  fall  into  two  groups.  Those  in  the  first 
group  are  mononuclear  and  non-granular.  Those  in 
the  second  group  are  polymorphonuclear  and  contain 
cytoplasmic  granules  which  are  distinguished  by  their 
size  and  staining  reactions.  In  its  structure  the  chief 
abnormal  leukocyte,  the  myelocyte,  combines  the  two 
groups,  being  mononuclear  like  the  first  and  granular 
like  the  second. 

The  leukocytic  percentages  given  here  as  normal  may 
be  taken  as  representing  about  the  average  for  this 
country.  Recent  studies  indicate  that  variations 
among  healthy  individuals  may  be  greater  than  has 
been  supposed  and  that  climatic  factors,  altitude,  etc., 
may  exert  a  decided  influence.  One  should  therefore 
hesitate  to  base  diagnostic  conclusions  upon  slight 
variations  in  the  differential  count  unless  one  has  previ- 
ously determined  the  normal  for  the  individual. 

(i)  Normal  Varieties. — (a)  Lymphocytes  are  small 
mononuclear  cells  without  specific  granules  (see  Front- 
ispiece and  Plate  IX).  They  are  about  the  size  of  a 
red  corpuscle  or  slightly  larger  (6-10  n),  and  consist  of 
a  single,  sharply  defined,  deeply  staining  nucleus,  sur- 
rounded by  a  narrow  rim  of  protoplasm.     The  nucleus 


STUDY    OF    STAINED   BLOOD  327 

is  generally  round,  but  is  sometimes  indented  at  one 
side.  Wright's  stain  gives  the  nucleus  a  deep  purple 
color  and  the  cytoplasm  a  pale  robin's-egg  blue  in 
typical  cells.  Larger  lymphocytes  are  frequently  found, 
especially  in  the  blood  of  children,  and  are  difficult  to 
distinguish  from  the  large  mononuclear  leukocytes.  It 
is  believed  that  the  larger  forms  are  young  lymphocytes, 
which  become  smaller  as  they  grow  older.  Some 
workers  record  the  large  and  small  lymphocytes  sepa- 
rately. There  is  no  clear  line  of  distinction,  but  if  it 
seems  desirable  to  separate  them,  the  terms  "imma- 
ture" and  "mature"  may  appropriately  be  used.  In 
the  cytoplasm  of  a  certain  percentage  of  lymphocytes 
the  Romanowsky  stains  show  a  variable  number  of 
reddish-purple  (azurophilic)  granules. 

Lymphocytes  are  formed  in  the  lymphoid  tissues, 
including  that  of  the  bone-marrow.  They  constitute 
about  25  to  33  per  cent,  of  all  leukocytes,  or  1200  to 
3300  per  cubic  millimeter  of  blood.  They  are  more 
abundant  in  the  blood  of  children,  averaging  about  60 
per  cent,  in  the  first  year  of  life  and  decreasing  to  about 
36  per  cent,  in  the  tenth,  the  immature  cells  being 
especially  abundant. 

The  percentage  of  lymphocytes  is  usually  moderately 
increased  in  those  conditions  which  give  leukopenia, 
especially  chlorosis,  pernicious  anemia,  and  many  de- 
bilitated conditions.  There  is  a  decided  absolute 
and  relative  increase  at  the  expense  of  the  poly- 
morphonuclears at  high  altitudes  although  the  extent 
of  this  is  somewhat  uncertain.  A  marked  increase, 
accompanied  by  an  increase  in  the  total  leukocyte 
count,  is  seen  in  pertussis  (Fig.    125)   and  lymphatic 


328  THE  BLOOD 

leukemia.  In  the  former  lymphocytes  average  about 
60  per  cent.  In  the  latter  they  sometimes  exceed  98 
per  cent.  Exophthalmic  goiter  commonly  gives  a 
marked  relative  lymphocytosis,  while  simple  goiter 
does  not  affect  the  lymphocytes.  In  pulmonary  tuber- 
culosis a  high  percentage  of  lymphocytes  or,  espe- 
cially, a  progressive  increase  is  a  favorable  prognostic 


Fig.   125. — Lymphocytosis,   case   of   pertusis   (  X    looo)    (courtesy  of 
Dr.  W.  P.  Harlow). 

sign,  while  a  progressive  decline  should  be  looked  upon 
with  apprehension.' 

There  is  at  present  a  tendency  toward  greater  con- 
servatism in  ascribing  diagnostic  significance  to  lympho- 
cytosis of  moderate  degree,  i.e.,  of  less  than  40  per 
cent.,  unless  the  normal  for  the  individual  has  been  pre- 
viously established.  Lymphocyte  percentages  as  low 
as  15  or  as  high  as  45  are  occasionally  met  with  in 
apparently  healthy  individuals. 


STUDY    OF    STAINED  BLOOD  329 

(b)  Large  Mononuclear  and  Transitional  Leuko- 
cytes (See  Frontispiece). — These  cells  are  two  or  three 
times  the  diameter  of  the  normal  red  corpuscle. 

The  large  mononuclear  contains  a  single  round  or 
oval  nucleus,  often  located  eccentrically.  The  zone  of 
protoplasm  surrounding  the  nucleus  is  relatively  wide. 
With  Wright's  stain  the  nucleus  is  less  deeply  colored 
than  that  of  the  lymphocyte,  while  the  cytoplasm  is 
very  pale  blue  or  colorless,  and  sometimes  contains  a 
few  reddish  granules.  The  size  of  the  cell,  the  width  of 
the  zone  of  cytoplasm,  and  the  depth  of  color  of  the 
nucleus  are  the  points  to  be  considered  in  distinguishing 
between  large  mononuclears  and  lymphocytes.  When 
large  forms  of  the  lymphocyte  are  present  the  distinc- 
tion is  often  difficult  or  impossible.  It  is  then  advisable 
to  count  the  two  cells  together  as  lymphocytes.  Some 
workers  arbitrarily  adopt  the  size  of  the  polymorpho- 
nuclear neutrophile  as  the  dividing  line  between  the 
two  cells. 

Transitional  leukocytes  are  simply  large  mononuclear 
leukocytes  whose  nuclei  are  lobulated,  deeply  indented 
or  horseshoe  shape.  There  is  no  good  reason  for  plac- 
ing them  in  a  separate  group  as  is  frequently  done. 
Mallory  and  others  class  the  two  cells  together  as 
** endothelial  leukocytes"  or  "endotheliocytes."  This 
is  convenient  but  it  seems  unwise  to  introduce  new 
names  until  the  nature  and  origin  of  the  cells  are  better 
understood. 

Comparatively  little  is  known  regarding  the  origin 
of  the  large  mononuclear  and  transitional  leukocytes. 
Some  at  least  appear  to  be  developed  from  the  endo- 
thelial cells  of  the  blood-  and  lymph-vessels  by  pro- 


330  THE  BLOOD 

liferation  and  desquamation.  Altogether  they  consti- 
tute 2  to  5  per  cent,  of  the  total  number  of  leukocytes; 
loo  to  600  per  cubic  millimeter  of  blood.  Only  a  few 
pathologic  conditions  raise  this  figure  to  any  marked 
degree.  A  distinct  increase  is  a  feature  of  the  blood 
in  typhoid  fever  and  may  be  of  some  value  in  differen- 
tial diagnosis.  It  is  also  quite  constant  in  malaria, 
where  sometimes  many  of  the  cells  contain  engulfed 
pigment  (see  Plate  VII).  Buntitig  has  found  it  also 
constant  early  in  Hodgkin's  disease  and  regards  it  as  an 
extremely  important  point  in  diagnosis.  Late  in  the 
disease  it  is  still  evident  but  is  then  overshadowed  by  an 
increase  of  neutrophiles. 

(c)  Polymorphonuclear  Neutrophilic  Leukocytes  (See 
Frontispiece). — There  is  usually  no  difficulty  in  recog- 
nizing these  cells.  Their  average  diameter  (about  12  ju) 
is  somewhat  less  than  that  of  the  large  mononu- 
clears. The  nucleus  stains  rather  deeply,  and  is  very 
irregular,  often  assuming  shapes  comparable  to  letters  of 
the  alphabet,  E,  Z,  S,  etc.  (Fig.  126).  Frequently  there 
appear  to  be  several  separate  nuclei,  hence  the  widely 
used  name,  "polynuclear  leukocyte."  Upon  careful  in- 
spection, however,  delicate  nuclear  bands  connecting 
the  parts  can  usually  be  seen.  The  cytoplasm  is  rela- 
tively abundant,  and  contains  great  numbers  of  very 
fine  neutrophilic  granules  (see  Fig.  130,  A).  With 
Wright's  stain  the  nucleus  is  bluish  purple,  and  the 
granules  reddish  lilac. 

Polymorphonuclear  leukocytes  are  formed  in  the 
bone-marrow  from  neutrophilic  myelocytes.  Ordinarily 
they  constitute  60  to  70  per  cent,  of  all  the  leukocytes : 
3000  to  7000  per  cubic  millimeter  of  blood.     An  occa- 


STUDY    OF   STAINED  BLOOD  33 1 

sional  normal  adult  may  give  a  count  as  low  as  40  per 
cent,  or  as  high  as  80  per  cent.  In  children  the  average 
runs  from  about  35  per  cent,  in  the  first  year  to  50  per 
cent,  in  the  tenth.  Any  marked  increase  in  their  num- 
ber practically  always  produces  an  increase  in  the  total 
leukocyte  count,  and  has  already  been  discussed  under 
Polymorphonuclear  Leukocytosis    (see   p.    289).     The 


Fig.   126. —  Marked     polymorphonuclear     neutrophilic     leukocytosis 
(  X  1000)  (courtesy  of  Dr.  W.  P.  Harlow). 

leukocytes  of  pus,  ptis-corpusdes,  belong  almost  wholly 
to  this  variety. 

A  comparison  of  the  percentage  of  polymorphonuclear 
cells  with  the  total  leukocyte  count  yields  more  informa- 
tion than  a  consideration  of  either  alone.  In  a  general 
way  the  percentage  represents  the  severity  of  the  infec- 
tion or,  more  correctly,  the  degree  of  toxic  absorption; 


332  THE  BLOOD 

while  the  total  count  indicates  the  patient's  power  of 
resistajice.  With  moderate  infection  and  good  resisting 
powers  the  leukocyte  count  and  the  percentage  of  poly- 
morphonuclears are  increased  proportionately.  When 
the  polymorphonuclear  percentage  is  increased  to  a 
notably  greater  extent  than  is  the  total  number  of  leuko- 
cytes, no  matter  how  low  the  count,  either  very  poor 
resistance  or  a  very  severe  infection  may  be  inferred. 

Gibson  has  suggested  the  use  of  a  chart  to  express  this 
relationship  graphically  (Fig.  127).  Its  arrangement  is 
purely  arbitrary,  but  it  will  be  found  very  helpful  in  in- 
terpreting counts.  An  ascending  line  from  left  to  right 
indicates  an  unfavorable  prognosis  in  proportion  as  the 
line  approaches  the  vertical.  All  fatal  cases  show  a  ris- 
ing line.  A  descending  or  horizontal  line  suggests  a  very 
favorable  prognosis. 

It  is  a  matter  of  observation  that  in  the  absence  of 
acute  infectious  disease  or  of  inflammation  directly  in 
the  blood  stream  (e.g.,  phlebitis,  sigmoid  sinusitis,  septic 
endocarditis),  a  polymorphonuclear  percentage  of  85  or 
over  points  very  strongly  to  gangrene  or  pus  formation 
somewhere  in  the  body.  On  the  other  hand,  excepting 
in  children,  where  the  percentage  is  normally  low,  pus 
is  uncommon  with  less  than  80  per  cent,  of  polymorpho- 
nuclears. 

Normally,  the  cytoplasm  of  leukocytes  stains  pale 
yellow  with  iodin.  Under  certain  pathologic  conditions 
the  cytoplasm  of  many  of  the  polymorphonuclears  stains 
diffusely  brown,  or  contains  granules  which  stain  reddish 
brown  with  iodin.  This  is  called  iodophilia.  Extracel- 
lular iodin-staining  granules,  which  are  present  nor- 
mally, are  more  numerous  in  iodophilia. 


STUDY    OF   STAINED  BLOOD 


333 


This  iodin  reaction  occurs  in  all  purulent  conditions 
except  abscesses  which  are  thoroughly  walled  off  and 


Total  leuko-  "Percentage  of 

cyte  count.  polymorphonuclears. 

Fig.  127. — Gibson  chart  with  blood-count  in  2  cases  of  appendicitis: 
Dotted  line  represents  a  mild  case  with  prompt  recovery;  the  continu- 
ous line,  a  very  virulent  streptococcic  case  with  poor  resistance,  peri- 
tonitis, and  early  death. 

purely  tuberculous  abscesses.  It  is  of  some  value  in 
diagnosis  between  serous  effusions  and  purulent  exu- 
dates, between  catarrhal  and  suppurative  processes  in 


334  THE   BLOOD 

the  appendix  and  Fallopian  tube,  etc.  Its  importance, 
however,  as  a  diagnostic  sign  of  suppuration  has  been 
much  exaggerated,  since  it  may  occur  in  any  general 
toxemia,  such  as  pneumonia,  influenza,  malignant  dis- 
ease, and  puerperal  sepsis. 

To  demonstrate  iodophilia,  place  the  air-dried  Jilms  in 
a  stoppered  bottle  containing  a  few  crystals  of  iodin  until 
the  films  become  yellow.  Mount  in  syrup  of  le\ailose 
and  examine  with  an  immersion  objective. 

Ameth's  Classification  of  Neutrophiles. — Arneth  groups 
the  neutrophilic  leukocytes  into  five  classes  according  to 
the  number  of  lobes  which  the  nucleus  possesses.  The 
forms  which  fall  into  each  class  and  the  average  normal 
percentages  as  given  by  Arneth  are  indicated  in  the  follow- 
ing list: 

Class  I.     One  round  or  indented  nucleus;  5  per  cent. 

Class  2.     Two  nuclear  divisions;  35  per  cent. 

Class  3.     Three  nuclear  divisions;  41  per  cent. 

Class  4.     Four  nuclear  divisions;  17  per  cent. 

Class  5.     Five  or  more  nuclear  divisions;  2  per  cent. 

This  is  really  a  classification  of  neutrophiles  according 
to  their  age,  the  youngest  cells  being  included  in  Class  i. 
Among  these  youngest  cells  are  the  myelocytes  and  meta- 
myelocytes which  do  not  appear  in  normal  blood. 

The  percentages  are  fairly  constant  in  the  same  individual 
in  health,  but  may  show  considerable  variations  in  disease, 
even  when  the  leukocyte  count  remains  unchanged.  An 
increase  of  the  lower  classes  at  the  expense  of  the  higher  is 
known  as  a  "shift  of  the  neutrophilic  blood  picture  to  the 
left."  The  opposite  condition  is  a  "shift  to  the  right." 
In  order  to  simplify  comparison  many  workers  in  this  country 
use  an  index  number  obtained  by  adding  the  first,  second, 
and  one-half  of  the  third  classes.     The  average  normal 


STUDY  OF  STAINED  BLOOD  335 

''Arneth  index"  is  accordingly  about  bo.  Briggs  found 
variations  between  51  and  65  in  normal  individuals. 

The  clinical  value  of  an  Arneth  count  is  not  definitely 
determined.  It  appears  to  have  greater  usefulness  in 
prognosis  than  in  diagnosis.  Most  pathologic  conditions 
which  produce  any  change  cause  a  shift  to  the  left,  i.e.,  a 
high  index.  Among  these  are  acute  infectious  diseases, 
pyogenic  infections  (appendicitis,  etc.),  and  tuberculosis. 
In  tuberculosis  the  Arneth  count  is  regarded  as  having  defi- 
nite prognostic  value,  the  higher  the  index  the  more 
serious  being  the  outlook. 

A  low  index  occurs  in  pernicious  anemia.  In  a  series  of 
23  examinations  in  12  cases  of  pernicious  anemia  Briggs 
found  an  average  index  of  40.29;  lowest,  16.5;  highest,  51.25. 
Eight  consecutive  cases  of  severe  secondary  anemia  (malig- 
nant disease,  syphilis,  nephritis,  repeated  hemorrhages, 
etc.)  gave  an  average  index  of  68.23,  only  one  case  (a 
case  of  syphilis  with  index  of  39)  falling  below  normal 
limits. 

For  the  Arneth  count  thin  well-stained  blood-films  are 
essential.  Wright's  stain  may  be  used  but  hematoxylin- 
eosin  is  better  since  it  brings  out  the  nuclear  structure 
more  clearly.  Nuclear  parts  which  are  joined  by  more 
than  a  thread  should  be  counted  as  one. 

Dohle's  Inclusion  Bodies. — In  191 1  Dohle  called  atten- 
tion to  the  occurrence  of  certain  peculiar  bodies  within  the 
cytoplasm  of  the  neutrophiles  in  cases  of  scarlet  fever 
(Fig.  128).  Their  nature  has  not  been  definitely  deter- 
mined. The  typical  "inclusion  bodies"  are  about  the  size 
of  micrococci  or  a  little  larger;  some  of  them  are  pear- 
shaped,  others  appear  like  short  rods  or  like  cocci  lying  in 
pairs.  Discrete,  punctiform  granules  are  sometimes  seen 
but  have  not  the  same  significance.  It  now  seems  well 
established  that  typical  inclusion  bodies  have  considerable 
diagnostic  value.     They  are  apparently  found  in  many  or 


336  THE  BLOOD 

even  the  majority  of  the  neutrophiUc  leukocytes  in  every 
case  of  scarlet  fever  early  in  the  disease.  Upon  the  other 
hand,  a  few  may  be  found  in  many  cases  of  diphtheria, 
pneumonia,  and  some  other  infectious  diseases,  but  never  in 
German  measles  and  rarely  in  measles. 

The  inclusion  bodies  can  be  seen  in  preparations  stained 
with  Wright's  stain,  but  long  staining  with  pyronin- 
methyl-green  is  preferable.  With  the  latter  stain,  nuclei 
are  purplish  and  the  bodies  bright  red. 

(d)  Eosinophilic  Leukocytes,  or  "Eosinophiles"  (See 
Frontispiece). — The  structure  of  these  cells  is  similar  to 
that  of  the  polymorphonuclear  neutrophiles,  with  the 


Fig.  128. — Dohle's  inclusion  bodies  in  leukocytes.  From  a  case  of 
scarlet  fever.  Pyronin-methyl-green  stain  (  X  1500)  (from  a  slide 
prepared  by  L.  W.  Hill). 

striking  difiference  that,  instead  of  fine  neutrophilic  gran- 
ules, their  cytoplasm  contains  coarse  round  or  oval  gran- 
ules having  a  strong  aflfinity  for  acid  stains.  They  are 
easily  recognized  by  the  size  and  color  of  the  granules, 
which  stain  bright  red  with  stains  containing  eosin  (see 
Fig.  130,  B).  Their  cytoplasm  has  generally  a  faint 
sky-blue  tinge,  and  the  nucleus  stains  somewhat  less 
deeply  than  that  of  the  polymorphonuclear  neutrophile. 
Eosinophiles  are  formed  in  the  bone-marrow  from 
eosinophilic  myelocytes.  Their  normal  number  varies 
from  50  to  400  per  cubic  millimeter  of  blood,  or  i  to  4 
per   cent,    of   the  leukocytes.     An   increase   is  called 


STUDY   OF   STAINED  BLOOD  337 

eosinophilia,  and  is  better  determined  by  the  actual 
number  than  by  the  percentage. 

Slight  eosinophilia  is  said  to  be  physiologic  during 
menstruation.  Marked  eosinophilia  is  always  patho- 
logic. It  occurs  in  a  variety  of  conditions,  the  most  im- 
portant of  which  are:  infection  by  animal  parasites; 
bronchial  asthma;  myelogenous  leukemia;  scarlet  fever; 
many  skin  diseases;  and  tuberculin  reactions. 

(a)  Eosinophilia  may  be  a  symptom  of  infection  by 
any  of  the  worms  and  from  a  diagnostic  view-point 
this  is  its  most  important  indication.  It  is  fairly  con- 
stant in  trichiniasis,  uncinariasis,  filariasis,  and  echino- 
coccus  disease,  and  is  usually  most  marked  in  the  first 
named  condition.  In  this  country  an  unexplained 
marked  eosinophilia  warrants  examination  of  a  portion 
of  muscle  for  Trichittella  spiralis  (see  p.  509).  The 
cells  usually  range  between  10  and  30  per  cent,  of  all 
the  leukocytes,  but  may  go  much  higher. 

{b)  True  bronchial  asthma  commonly  gives  a  marked 
eosinophilia  during  and  following  the  paroxysms.  This 
is  helpful  in  excluding  asthma  of  other  origin.  Eosino- 
philes  also  appear  in  the  sputum  in  large  numbers. 

(c)  In  myelogenous  leukemia  there  is  almost  invariably 
an  absolute  increase  of  eosinophiles,  although,  owing  to 
the  great  increase  of  other  leukocytes,  the  percentage  is 
usually  diminished.  Dwarf  and  giant  forms  are  often 
numerous. 

{d)  Scarlet  fever  is  frequently  accompanied  by  eosino- 
philia, which  may  help  to  distinguish  it  from  measles. 

(e)  Eosinophilia  has  been  observed  in  a  large  number 
of  skin  diseases,  notably  pemphigus,  prurigo,  psoriasis, 


22 


338  THE    BLOOD 

and  urticaria.  It  probably  depends  less  upon  the  variety 
of  the  disease  than  upon  its  extent. 

if)  Eosinophilic  cells  are  usually  increased  to  a  vari- 
able degree  in  tuberculin  reactions  and  anaphylactic 
conditions  in  general. 

(e)  Basophilic  Leukocytes  or  "Mast-cells"  (See  Fron- 
tispiece).— In  general,  these  resemble  polymorphonu- 
clear neutrophiles  except  that  the  nucleus  is  less  irregular 
(usually  merely  indented  or  slightly  lobulated)  and  that 


Fig.  129. — Basophilic  leiikoc> 

undergoing  mitosis  (  X  1000;. 

the  granules  are  larger  and  have  a  strong  affinity  for 
basic  stains.  They  are  easDy  recognized  (Figs.  129  and 
130,  C),  Sometimes  one  sees  cells  from  which  most  of 
the  granules  have  disappeared,  leaving  clean-cut  open- 
ings. With  Wright's  stain  the  granules  are  deep  pur- 
ple, while  the  nucleus  is  pale  blue  and  is  often  nearly 
or  quite  hidden  by  the  granules,  so  that  its  form  is 
difficult  to  make  out.  Basophilic  granules  are  not  col- 
ored by  Ehrlich's  stain. 

Mast-cells  probably  originate  in  the  bone-marrow  from 


STUDY  or  STAINED  BLOOD 


339 


basophilic  myelo<:\i;es.     They  are  least  numerous  of  the 
loikocytes  in  normal  blood,  rarely  exceeding  0.5  per 


B  C 

i  lexikacjtes,  sbamissfi  tsiaiivK  shut  o£  granules:  A. 
.:  B,  e«]sinofildSc;  C.  baaopliiBe  (X  looo). 

cent.,  or  25  to  50  per  cubic  millimeter.     A  notable 
is  limited  almost  exclusively  to  myelogenous 
here  they  are  sometimes  ver\'  numeioas. 


Fig.  131. —  - 


f  2>  Abnormal  Varieties. — iji)  Myelocytes  (see  Fron- 
md  Fig.  131)  are  large  mononuclear  cells  whose 


340  THE   BLOOD 

cytoplasm  is  filled  with  granules.  Typically,  the  nucleus 
occupies  about  one-half  of  the  cell,  and  is  round  or  oval, 
or  is  indented,  with  its  convex  side  in  contact 
with  the  periphery  of  the  cell.  It  stains  rather  feebly. 
The  average  diameter  of  this  cell  (about  15.75  ^i)  is 
greater  than  that  of  any  other  leukocyte,  but  there  is 
much  variation  in  size  among  individual  cells.  Myelo- 
cytes are  named  according  to  the  character  of  their 
granules — neutrophilic,  eosinophilic,  and  basophilic 
myelocytes.  These  granules  are  identical  with  the  cor- 
responding granules  in  the  leukocytes  just  described. 
They  are,  however,  often  less  distinct  and  less  sharply 
differentiated  by  the  various  stains  than  those  of  the 
corresponding  polymorphonuclear  cells.  In  some  the 
granules  are  few  in  number,  the  cells  departing  but 
little  from  the  structure  of  the  parent  myeloblast. 
Such  cells  may  be  called  "premyelocytes."  In  young 
neutrophilic  myelocytes  there  is  a  tendency  to  rela- 
tively large  granules  which  take  a  purple  color  with 
Wright's  stain.  Although  the  occurrence  of  two  kinds 
of  granules  in  the  same  cell  is  rare,  a  few  basophilic 
granules  are  sometimes  seen  in  young  eosinophilic  mye- 
locytes. The  basophilic  myelocyte  is  usually  small; 
and  its  nucleus  is  commonly  so  pale  and  so  obscured 
by  the  granules  that  the  cell  is  not  easily  distinguished 
from  the  mast-cell. 

The  small  neutrophilic  cell  with  a  single  small  round 
deeply  staining  nucleus  which  is  sometimes  encountered 
must  not  be  confused  with  the  myelocyte.  Such  atypic 
cells  probably  result  from  division  of  polymorphonu- 
clear neutrophiles. 

Myelocytes  are  the  bone-marrow  cells  from  which 


STUDY    OF    STAINED  BLOOD  34I 

the  corresponding  granular  leukocytes  are  developed. 
They  in  turn  are  derived  from  certain  non-granular  cells 
of  the  bone-marrow,  the  myeloblasts.  Their  presence 
in  the  blood  in  considerable  numbers  is  diagnostic  of 
myelogenous  leukemia.  The  neutrophilic  form  is  the 
least  significant.  A  few  of  these  may  be  present  in  very 
marked  leukocytosis  or  any  severe  blood  condition,  as 
pernicious  anemia.  In  the  anemia  of  malignant  dis- 
ease they  suggest  bone-marrow  metastasis.  Eosino- 
philic myelocytes  are  found  only  in  myelogenous  leu- 
kemia,  where   they   are   often   very   numerous.     The 


Fig.  132. — A  myeloblast  and  a  neutrophilic  leukocyte.     From  a  case  of 
myelogenous  leukemia.     Wright's  stain  (  X  lodo). 

basophilic  variety  is  less  common,  and  is  confined  to 
long-standing,  severe  myelogenous  leukemia. 

(b)  Myeloblasts. — These  are  the  parent  cells  of  the 
myelocytes,  from  which  they  differ  chiefly  in  the  ab- 
sence of  cytoplasmic  granules.  Their  round  or  oval 
nuclei  are  poor  in  chromatin  and  contain  several  rather 
indistinct  nucleoli  (Fig.  132).  The  cytoplasm,  which 
is  generally  not  abundant,  is  markedly  basophilic, 
staining  pure  blue  with  Wright's  stain.  In  some 
preparations  it  is  characteristically  smooth  in  texture; 
in  others  it  is  finely  reticular. 


342  THE   BLOOD 

Myeloblasts  appear  in  the  blood  in  large  numbers  in 
acute  myelogenous  leukemia  and  the  terminal  stages 
of  chronic  myelogenous  leukemia,  when  the  bone- 
marrow  reverts  to  the  embryonic  type.  Their  number 
is  therefore  important  in  prognosis.  They  may  be  in- 
distinguishable morphologically  from  the  large  lympho- 
cytes of  acute  lymphatic  leukemia,  but  can  usually  be 
distinguished  by  the  oxydase  reaction.  In  most  ad- 
vanced cases  of  myelogenous  leukemia  all  stages  of 
transition  between  the  myeloblast  and  myelocyte  may 
be  found. 

Indophenol  Oxydase  Test. — The  technic  used  by  Evans 
is  as  follows: 

1.  Fix  cover-glass  films  eight  hours  in  formaldehyd  vapor 
in  a  closed  jar. 

2.  Stain  eight  minutes  with  a  saturated  aqueous  solution 
of  safranin  or  2  per  cent,  solution  of  pyronin. 

3.  Wash  quickly  in  water  and  blot  dry. 

4.  Upon  a  slide  mix  i  drop  of  a  i  per  cent,  aqueous 
solution  of  dimethylparaphenylendiamin  (less  than  three 
weeks  old)  and  i  drop  of  a  i  per  cent,  solution  of  alpha- 
naphthol  in  i  per  cent,  potassium  hydroxid  (less  than  four 
days  old).  Upon  this  place  the  previously  stained  cover, 
film-side  down,  and  examine  immediately. 

In  such  preparations  the  nuclei  show  a  pink  or  orange 
color  which  fades  after  a  few  minutes.  The  cytoplasm 
of  cells  containing  oxydase — polymorphonuclears,  large 
mononuclears  and  transitionals,  myelocytes,  and  myelo- 
blasts— gradually  becomes  a  faint  diffuse  blue,  and  fine  and 
coarse  blue-black  granules  appear.  The  reaction  endures 
about  ten  minutes.  Lymphocytes,  red  corpuscles,  and 
platelets  should  show  no  blue. 


STUDY    OF    STAINED   BLOOD  343 

ic)  Tiirck's  Irritation  Leukocytes.— Strictly  speak- 
ing these  ought  to  be  classed  with  the  normal  leuko- 
cytes since  they  are  often  encountered  in  normal  blood, 
where,  however,  their  number  rarely  exceeds  i  per  cent, 
of  the  leukocytes.  They  are  generally  included  with 
the  large  mononuclear  leukocytes.  In  brief  they  are 
large,  mononuclear,  non-granular  cells  with  dense, 
opaque  cytoplasm  which  stains  deep  blue  with  Wright's 
stain  and  brown  with  Ehrlich's  and  sometimes  contains 
vacuoles  (see  Plates  I  and  VIII).  As  a  rule  they  are 
not  difficult  to  recognize  although  they  might  be  con- 
fused with  the  large  lymphocytes  or  with  very  strongly 
polychromatophilic  megaloblasts. 

At  present  Tiirck's  irritation  leukocytes  have  no 
diagnostic  importance.  Considerable  numbers  may 
appear  in  the  blood  in  conditions  associated  with  irrita- 
tion of  the  bone-marrow,  notably  primary  and  second- 
ary anemia,  leukemia  and  malaria. 

(d)  Degenerated  Forms. — These  are  frequently  met 
but  have  no  significance  unless  present  in  large  numbers. 
They  include  (a)  vacuolated  leukocytes  and  (b)  bare 
nuclei  from  ruptured  cells.  The  former  are  found 
most  frequently  in  toxemias  and  leukemia.  A  few  of 
the  latter  are  present  in  every  blood  smear  but  are 
especially  abundant  in  leukemia  (Fig.  133).  They  vary 
from  fairly  well  preserved  nuclei  to  mere  strands  of 
palely  stained  nuclear  substance  arranged  in  a  coarse 
network — the  so-called ' '  basket-cells ' '  (see  Frontispiece) . 

Occasionally  in  lymphatic  leukemia  frayed-out  nuclei 
without  cytoplasm  exceed  the  usual  lymphocytes  in 
number.  In  such  cases  some  writers  infer  involvement 
of   the  bone-marrow,   holding  that  the  naked  nuclei 


344  THE   BLOOD 

represent  very  fragile  bone-marrow  cells  which  have 
gone  to  pieces  in  the  circulation.  In  many  cases  at 
least  it  seems  more  likely  that  such  nuclei  only  repre- 
sent fragile  lymphocytes  which  have  been  broken  in 
making  the  smear. 

(e)  Atypic  Forms. — ^Leukocytes  which  do  not  lit  in 
with  the  above  classification  are  not  infrequently  met, 
especially  in  high-grade  leukocytosis,  pernicious  anemia, 
and  leukemia.  They  are  always  more  abundant  in 
childhood.     The  nature  of  many  of  them  is  not  clear, 


Fig.  133. — Blood  in  chronic  lymphatic  leukemia,  showing  many  rup- 
tured lymphocytes  (  X  750). 

and  their  number  is  usually  so  small  that  they  may  be 
classed  as  "undetermined"  in  making  a  differential 
count. 

3.  Blood=plateIets. — These  are  not  colored  by  Ehr- 
lich's  stain  nor  by  hematoxylin  and  eosin.  With 
Wright's  stain  they  appear  as  spheric  or  ovoid,  reddish 
to  violet,  granular  bodies,  2  to  4  /i  in  diameter.  Occa- 
sionally a  platelet  as  large  as  a  red  corpuscle  is  seen. 
When  well  stained  a  delicate  hyaline  peripheral  zone 


BLOOD   PARASITES  345 

can  be  distinguished.  In  ordinary  blood-smears  they 
are  usually  clumped  in  masses.  A  single  platelet  lying 
upon  a  red  corpuscle  may  easily  be  mistaken  for  a 
malarial  parasite  (see  Frontispiece  and  Fig.  134). 

Blood-platelets  are  being  much  studied  at  present, 
but,  aside  from  the  facts  mentioned  under  their  enum- 
eration (see  p.  300),  little  of  clinical  value  has  been 
learned.  They  have  been  variously  regarded  as  very 
young  red  corpuscles  (the  "hematoblasts"  of  Hay  em) 


Fig.   134. — A  cluster  of  blood-platelets  and  two  platelets  lying  upon  a 
red  cell  and  simulating  malarial  parasites  (  X  looo). 

as  disintegration  products  of  leukocytes,  as  remnants 
of  extruded  nuclei  of  erythrocytes,  and  as  independent 
nucleated  bodies.  The  most  probable  explanation  of 
their  origin  seems  to  be  that  of  J.  H.  Wright,  who  re- 
gards them  as  detached  portions  of  the  cytoplasm  of 
certain  giant-cells  of  the  bone-marrow  and  spleen. 

X.  BLOOD  PARASITES 
A.  Bacteria 

Bacteriologic  study  of  the  blood  is  useful  in  many 
conditions,    but,   in   general,    the  somewhat  elaborate 


346  THE  BLOOD 

technic  involved  takes  it  out  of  reach  of  the  clinician. 
As  applied  to  the  diagnosis  of  typhoid  fever,  however, 
the  technic  of  blood- cultures  has  been  so  simplified  that 
it  can  be  carried  through  by  any  one  who  is  competent 
to  do  the  simplest  cultural  work. 

Typhoid  bacilli  can  be  detected  in  the  blood  in  prac- 
tically every  case  of  typhoid  fever  in  the  first  week  of 
the  disease;  in  about  80  to  85  per  cent,  of  cases  in  the 
second  week;  and  in  decreasing  percentages  in  the  later 
weeks.  The  blood-culture,  therefore,  offers  the  most 
certain  means  of  early  diagnosis.  It  is  in  a  sense  com- 
plementary to  the  Widal  reaction,  the  former  decreasing 
and  the  latter  increasing  in  reliability  as  the  disease 
progresses.  The  blood-culture  gives  best  results  before 
the  Widal  appears,  as  one  would  expect  from  the  fact 
that  the  Widal  test  depends  upon  the  presence  of  anti- 
bodies which  destroy  or,  at  least,  injure  the  bacilh. 
The  two  methods  together  will  establish  the  diagnosis 
in  practically  every  case  at  any  stage.  Bacilli  disap- 
pear from  the  blood  in  convalescence  and  reappear  in 
a  relapse. 

Technic  of  Blood-cultures  in  Typhoid  Fever. — The  blood 
may  be  obtained  in  one  of  two  ways: 

(a)  With  a  spring-lancet  (see  Fig.  85)  make  a  deep  punc- 
ture in  the  edge  (not  the  side)  of  the  lobe  of  the  ear,  as  for  a 
blood-count.  Allow  the  blood  to  drop  directly  into  a  short 
culture-tube  containing  the  bile  medium.  By  gentle 
milking,  20  to  40  drops  can  usually  be  obtained.  This 
simple  method  of  obtaining  blood  is  especially  applicable 
during  the  first  week  of  the  disease  when  bacilli  are  abun- 
dant. Contamination  with  skin  cocci  is  possible,  but  does 
not  usually  interfere  when  the  bile  medium  is  used. 


BLOOD    PARASITES  347^ 

(b)  In  the  later  weeks  of  the  disease  a  larger  quantity  of 
blood  is  needed  and  must  be  obtained  from  a  vein  as  de- 
scribed on  page  254. 

As  special  culture-medium,  ox-bile  is  generally  used.  It 
favors  the  growth  of  the  typhoid  bacillus  and  retards  the 
growth  of  other  organisms.  A  good  formula  is  given  on 
page  569. 

As  soon  as  convenient  after  the  blood  is  added,  place  the 
tubes  in  the  incubator.  After  about  twelve  hours  examine 
for  motile  bacilli.  If  none  are  found,  transfer  a  few  drops 
to  tubes  of  bouillon  or  solidified  blood-serum  and  incubate 
for  twelve  hours  longer.  If  motile,  Gram-negative  bacilli 
are  found,  they  are  almost  certainly  typhoid  bacilli. 
Further  study  is,  however,  desirable,  especially  from  a 
scientific  point  of  view.  The  only  bacilli  which  might  cause 
confusion  are  the  paratyphoid  and  colon  bacilli,  which  can 
be  distinguished  by  gas  production  in  glucose  media,  indol 
production,  and  their  effect  upon  litmus  milk  (see  p.  583). 
The  agglutination  test  for  the  identity  of  the  bacillus  is  not 
available  clinically,  since  freshly  isolated  bacilli  do  not 
agglutinate  well. 

Technic  for  Other  Bacteria. — About  lo  c.c.  of  blood  are 
obtained  from  a  vein  (see  p.  254)  under  strictly  aseptic 
precautions  and  immediately  distributed  among  flasks  of 
sterile  bouillon.  When  the  pneumococcus  or  streptococcus 
is  suspected  a  better  medium  is  nutrient  bouillon  to  which 
one-fourth  its  volume  of  sterile  ascitic  fluid  has  been  added. 
Ordinarily  not  more  than  i  c.c.  of  blood  should  be  added  to 
50  or  100  c.c.  of  culture  medium.  After  incubating  for 
twenty-four  hours  or  longer,  sub-cultures  are  made  from 
these  flasks  upon  media  appropriate  to  identify  any  bacteria 
which  may  grow. 

When  the  blood  must  be  obtained  at  a  distance  from  the 
laboratory  it  may  be  received,  by  means  of  one  of  the  de- 
vices shown  in  Figs.  87  and  89,  directly  into  15  c.c.  of  a 


348  THE  BLOOD 

sterile  solution  consisting  of  2  Gm.  of  ammonium  oxalate 
and  6  Gm.  sodium  chlorid  in  1000  c.c.  distilled  water. 
Such  a  solution  will  prevent  coagulation  and  will  not  harm 
any  bacteria  that  may  be  present.  As  soon  as  convenient 
the  blood  is  added  to  appropriate  culture  media  in  flasks 
or  tubes,  or  is  mixed  with  melted  agar  and  poured  into 
Petri  plates. 

B.  Animal  Parasites 

Of  the  animal  parasites  which  have  been  found  in  the 
blood,  five  are  interesting  clinically:  the  spirochete  of 
relapsing  fever;  trypanosomes;  malarial  parasites;  fila- 
rial larvae;  and  the  larvae  of  Trichinella  spiralis. 

1 .  Spirpchaeta  recurrentis  is  described  on  page  460. 

2.  Trypanosoma  gambiense. — Various  trypano- 
somes are  common  in  the  blood  of  fishes,  amphibians, 
birds,  and  mammals  (see  Fig.  159).  They  live  in  the 
blood-plasma  and  do  not  attack  the  corpuscles.  In 
some  animals  they  are  apparently  harmless;  in  others 
they  are  an  important  cause  of  disease.  They  are 
discussed  more  fully  on  page  464. 

The  trypanosome  of  human  blood,  Trypanosoma  gam- 
biense (Plate  VI),  is  an  actively  motile,  spindle-shaped 
organism,  two  or  three  times  the  diameter  of  a  red  cor- 
puscle in  length,  with  an  undulating  membrane  which 
terminates  at  the  anterior  end  in  a  long  flagellum.  It 
can  be  seen  with  medium-power  objectives  in  fresh 
blood,  but  is  best  studied  with  an  oil-immersion  lens  in 
preparations  stained  as  for  the  malarial  parasite.  It 
will  be  necessary  to  search  many  slides.  The  concen- 
tration method  described  for  the  larvae  of  Trichinella 
(see  p.  363)  may  be  used.  Human  trypanosomiasis  is 
common  in  Africa.     As  a  rule,  it  is   a   very  chronic 


PLATE  VI 


f fc.!4 


«*■ 


1 


Trypanosoma  gambiense. 


Half-grown     tertian      malarial  Estivo-autumnal  malarial  par- 

parasites  in  stippled   cells  and  a       asites,  small  ring  forms  and  cres- 
group  of  spores  from  a  freshly  rup-       cents, 
tured  scgmenter.     From  a  slide  of 
double    tertian    malarial    concen- 
trated by  F.  M.  Johns. 


■  1^  _  dB      ^BIm    jtffll     ^P^^ 


Spirochetes  in  the  blood  of  a  case  of  relapsing  fever  originating  in  Colo- 
rado.     Reported  by  Dr.  C.  N.  Meader. 


BLOOD   PARASITES  349 

disease.  "Sleeping  sickness"  is  a  late  stage  when  the 
organisms  have  invaded  the  cerebrospinal  fluid.  Infec- 
tion is  carried  by  the  tsetse  fly,  Glossina  palpalis. 

3.  The  Malarial  Parasites. — These  protozoa  belong 
to  the  Sporozoa  (see  p.  470),  order  Hemosporidia,  the 
members  of  which  are  parasites  in  the  blood  of  a  great 
variety  of  vertebrates.  Three  species,  constituting  the 
genus  Plasmodium,  are  associated  with  malarial  fever  in 
man:  Plasmodium  vivax,  P .  malarice,  and  P.  falciparum, 
the  parasites  respectively  of  the  tertian,  quartan,  and 
estivo-autumnal  types  of  malaria.  The  life  histories  of 
the  three  are  so  similar  that  they  may  well  be  described 
together, 

(i)  Life  Histories. — There  are  two  cycles  of  develop- 
ment: one,  the  asexual,  in  the  blood  of  man;  and  the 
other,  the  sexual,  in  the  intestinal  tract  of  a  particular 
genus  of  mosquito,  Anopheles. 

(a)  Asexual  Cycle. — The  young  organism  enters  the 
blood  through  the  bite  of  the  mosquito.  It  makes  its 
way  into  a  red  corpuscle,^  where  it  appears  as  a  small, 
pale,  "hyaline"  body.  This  body  exhibits  ameboid 
movement  and  increases  in  size.  Soon  dark-brown 
granules,  derived  from  the  hemoglobin  of  the  corpuscle, 
make  their  appearance  within  it.  When  it  has  reached 
its  full  size — filling  and  distending  the  corpuscle  in  the 
case  of  the  tertian  parasite,  smaller  in  the  others — the 

^In  this  section  the  malarial  parasite  is  described,  in  accordance 
with  the  usual  teaching,  as  living  within  the  parasitized  red  corpuscle. 
The  recent  work  of  Mary  Rowley-Law^son,  however,  tends  to  show 
that  the  parasite  is  extra-cellular  throughout  its  whole  existence;  that 
it  attaches  itself  to  the  external  surface  of  the  red  corpuscle  but  does 
not  enter  it;  and  that  it  migrates  from  corpuscle  to  corpuscle  between 
paroxysms,  destroying  each  cell  before  it  abandons  it. 


35©  THE  BLOOD 

pigment  granules  gather  at  the  center  or  at  one  side;  the 
organism  divides  into  a  number  of  small  hyaline  bodies, 
the  spores  or  merozoites;  and  the  red  corpuscle  bursts, 
setting  spores  and  pigment  free  in  the  blood-plasma. 
This  is  called  segmentation.  It  coincides  with,  and  by 
liberation  of  toxins  causes,  the  paroxysm  of  the  disease. 
A  considerable  number  of  the  spores  are  destroyed  by 
leukocytes  or  other  agencies;  the  remainder  enter  other 
corpuscles  and  repeat  the  cycle.  Many  of  the  pigment 
granules  are  taken  up  by  leukocytes.  In  estivo-autum- 
nal  fever  segmentation  occurs  in  the  internal  organs  and 
the  segmenting  and  larger  pigmented  forms  are  seldom, 
seen  in  the  peripheral  blood.  This  accumulation  of 
parasites  in  the  internal  organs  explains  certain  tj'pes  of 
pernicious  estivo-autumnal  malaria,  e.g.,  the  comatose 
type,  when  the  parasites  accumulate  in  the  capillaries 
of  the  brain. 

The  asexual  cycle  of  the  tertian  organism  occupies 
forty-eight  hours;  of  the  quartan,  seventy-two  hours; 
of  the  estivo-autumnal,  an  indefinite  time — usually 
twenty-four  to  forty-eight  hours. 

The  parasites  are  thus  present  in  the  blood  in  great 
groups,  all  the  individuals  of  which  reach  maturity  and 
segment  at  approximately  the  same  time.  This  ex- 
plains the  regular  recurrence  of  the  paroxysms  at  inter- 
vals corresponding  to  the  time  occupied  by  the  asexual 
cycle  of  the  parasite.  Not  infrequently  there  is  mul- 
tiple infection,  one  group  reaching  maturity  while  the 
others  are  still  young;  but  the  presence  of  two  groups 
which  segment  upon  the  same  day  is  extremely  rare. 
Fevers  of  longer  intervals — six,  eight,  ten  days — are  prob- 
ably due  to  the  ability  of  the  body,  sometimes  of  itself, 


BLOOD   PARASITES  35 1 

sometimes  by  aid  of  quinin,  to  resist  the  parasites,  so 
that  numbers  sufficient  to  cause  a  paroxysm  do  not 
accumulate  in  the  blood  until  after  several  repetitions 
of  the  asexual  cycle.  In  estivo-autumnal  fever  the 
regular  grouping,  while  usually  present  at  first,  is  soon 
lost,  thus  causing  "irregular  malaria," 

Bass  has  recently  succeeded  in  cultivating  the  mala- 
rial parasite  outside  of  the  body. 

(b)  Sexual  Cycle. — Besides  the  ameboid  individuals 
which  pass  through  the  asexual  cycle,  there  are  present 
with  them  in  the  blood  many  individuals  with  sexual 
properties.  These  are  called  gametes.  The  female  is 
a  little  the  larger.  The  gametes  do  not  undergo  seg- 
mentation, but  grow  to  adult  size  and  remain  inactive 
in  the  blood  until  taken  up  by  a  mosquito.  Many  of 
them  are  apparently  extracellular,  but  stained  prepara- 
tions usually  show  them  to  be  surrounded  by  or 
attached  to  the  remains  of  a  corpuscle.  In  tertian 
and  quartan  malaria  they  resemble  the  asexual 
individuals  until  a  variable  time  after  the  blood  leaves 
the  body,  when  the  male  gamete  sends  out  one  or  more 
flagella.  In  estivo-autumnal  malaria  the  gametes  take 
distinctive  ovoid  and  crescentic  forms,  and  are  not 
difficult  to  recognize.  These  sexual  forms  are  very  re- 
sistant to  quinin  and  often  persist  in  the  blood  long  after 
the  ameboid  forms  have  been  destroyed.  Under  ordi- 
nary conditions  they  are  incapable  of  continuing  the 
disease  until  they  have  passed  through  the  cycle  in  the 
mosquito,  but  it  seems  probable  that  under  certain 
unusual  conditions  the  female  gamete  may,  without 
fertilization,  undergo  further  development  and  sporulate, 
thus  starting  a  new  asexual  cycle. 


352 


THE  BLOOD 


When  a  malarious  person  is  bitten  by  a  mosquito,  the 
gametes  are  taken  with  the  blood  into  its  stomach. 
Here  the  male  sends  out  one  or  more  flagella.  These 
break  off  and  seek  out  the  females,  whom  they  fertil- 
ize much  as  the  sperm  fertilizes  the  o\Tim.  The  female 
soon  thereafter  becomes  encysted  in  the  wall  of  the  intes- 
tine. After  a  time  this  "oocyst"  ruptures,  liberating 
many  minute  rods,  or  sporozoites,  which  have  formed 


Male.  Female. 

Fig.  135. — Head  of  Culex  (after  Giles).  Showing  the  straight 
proboscis,  the  jointed  palpi  and,  external  to  these,  the  hairy  an- 
tennae. The  male  is  distinguished  from  the  female  by  the  longer 
hairs  on  the  antennae.  Note  that  the  palpi  of  the  male  are  longer 
than  the  proboscis,  while  those  of  the  female  are  very  much  shorter 
(compare  with  Fig.  136). 

within  it.  These  migrate  to  the  salivary  glands,  and  are 
carried  into  the  blood  of  the  person  whom  the  mosquito 
bites.  Here  they  enter  red  corpuscles  as  young  mala- 
rial parasites,  and  the  majority  pass  through  the  asexual 
cycle  just  described. 

The  sexual  cycle  can  take  place  only  within  the  body 
of  the  female  of  one  genus  of  mosquito,  Anopheles. 
The  male  does  not  bite.     Absence  of  this  mosquito  from 


BLOOD    PARASITES 


353 


certain  districts  explains  the  absence  of  malaria.  It  is 
distinguished  from  our  common  house  mosquito,  Culex, 
by  the  relative  lengths  of  proboscis  and  palpi  (Figs.  135 
and  136),  which  can  be  seen  with  a  hand-lens,  by  its 
attitude  when  resting,  and  by  its  dappled  wing  (Fig. 
137).  Anopheles  is  strictly  nocturnal  in  its  habits;  it 
usually  flies  low,  and  rarely  travels  more  than  a  few 
hundred  yards  from  its  breeding-place,  although  it  may 


Male.  Female. 

Fig.  136. — Head  of  Anopheles  (after  Giles).  The  sexes  are  distin- 
guished by  the  antennae  as  noted  under  Fig.  135.  In  this  mosquito 
the  palpi  of  both  sexes  are  nearly  the  same  length  as  the  proboscis. 

be  carried  by  winds .  These  facts  explain  certain  peculi- 
arities in  malarial  infection;  thus,  infection  occurs  prac- 
tically only  at  night;  it  is  most  common  near  stagnant 
water,  especially  upon  the  side  toward  which  the  pre- 
vailing winds  blow;  and  the  danger  is  greater  when 
persons  sleep  upon  or  near  the  ground  than  in  upper 
stories  of  buildings.  The  insects  frequently  hibernate 
in  warmed  houses,  and  may  bite  during  the  winter. 
A  mosquito  becomes   dangerous  in  eight  to  fourteen 

23 


354 


THE   BLOOD 


days  after  it  bites  a  malarious  person,  and  remains  so 
throughout  its  life. 

(2)  Detection. — Search  for  the  malarial  parasite  may- 
be made  in  either  fresh  blood  or  stained  films.     If  pos- 


FiG.  137. — Showing,  on  the  left.  Anopheles  in  resting  position,  its 
dappled  wing,  and  the  position  of  its  larvae  in  water;  on  the  right, 
Culex  in  resting  position,  its  plain  wing,  and  the  position  of  its  larvae  in 
water.  The  arrows  indicate  the  directions  taken  by  the  larvae  when 
the  water  is  disturbed  (Abbott). 


sible,  the  blood  should  be  obtained  a  few  hours  before 
the  chill — not  during  it  nor  within  a  few  hours  after- 
ward, since  at  that  time  (in  single  infections)  only  the 
very  young,  unpigmented  forms  are  present,  and  these 
are  the  most  difficult  to  find  and  recognize.     Sometimes 


BLOOD    PARASITES  355 

many  parasites  are  found  in  a  microscopic  field;  some- 
times, especially  in  estivo-autumnal  infection,  owing  to 
accumulation  in  internal  organs,  careful  search  is  re- 
quired to  find  any,  despite  very  severe  symptoms. 
Quinin  causes  them  rapidly  to  disappear  from  the 
peripheral  blood,  and  few  or  none  may  be  found  after 
its  administration.  In  the  absence  of  organisms,  the 
presence  of  pigment  granules  within  leukocytes — espe- 
cially the  large  mononuclears — may  be  taken  as  pre- 
sumptive evidence  of  malaria.  Pigmented  leukocytes 
(see  Frontispiece  and  Plate  VII)  are  most  numerous 
after  a  paroxysm. 

(a)  In  Fresh  Unstained  Blood.- — Obtain  a  small  drop 
of  blood  from  the  finger  or  lobe  of  the  ear.  Touch 
the  center  of  a  cover-glass  to  the  top  of  the  drop  and 
quickly  place  it,  blood  side  down,  upon  a  slide.  If 
the  slide  and  cover  be  perfectly  clean  and  the  drop  not 
too  large,  the  blood  will  spread  out  so  as  to  present  only 
one  layer  of  corpuscles.  Search  with  an  oil-immersion 
objective,  using  very  subdued  light.  The  preparation 
may  be  kept  for  many  hours  if  ringed  with  vaselin  or 
melted  paraffin 

The  young  organisms  appear  as  small,  round,  ring- 
like or  irregular,  colorless  bodies  within  red  corpuscles. 
The  light  spots  caused  by  crenation  and  other  changes 
in  the  corpuscles  are  frequently  mistaken  for  them,  but 
are  generally  more  refractive  or  have  more  sharply  de- 
fined edges.  The  older  forms  are  larger  colorless  bodies 
containing  granules  of  brown  pigment.  In  the  case  of 
the  tertian  parasite,  these  granules  have  active  vibra- 
tory motion,  which  renders  them  conspicuous;  and  as 
the  parasite  itself  is  very  pale,  one  may  see  only  a  large 


356 


THE  BLOOD 


pale  corpuscle  in  which  fine  pigment  granules  are  danc- 
ing. Segmenting  organisms,  when  typic,  appear  as 
rosets,  often  compared  to  daisies,  the  petals  of  which 
represent  the  segments,  while  the  central  brown  por- 
tion represents  the  pigment.  Tertian  segmenting 
forms  are  less  frequently  typic  than  quartan.  Flagel- 
lated forms  are  not  seen  until  ten  to  twenty  minutes 
after  the  blood  has  left  the  vessels.     As  Cabot  suggests, 


VARIETIES   OF   THE   MALARIAL   ORGANISM 


Tertian. 

Quartan. 

ESTIVO-ADTUMNAL. 

Asexual  cycle,  forty-eight 
hours. 

Seventy-two  hours. 

Usually  twenty-four  to 
forty-eight  hours. 

Substance  pale,  trans- 
parent,   comparable    to 
hyaline  tube-cast. 

Outline  indistinct. 

Ameboid  motion  ac- 
tive. 

Mature  asexual  form 
large;  fills  and  often  dis- 
tends corpuscle. 

Pigment  -granules 
fine,    brown,    scattered 
throughout.      Very   ac- 
tive dancing  motion. 

Segmenting  body 
rarely   assumes   typical 
"daisy"  form.      15    to 
20  segments. 

Gametes  resemble 
asexual  forms. 

Red   corpuscles  pale 
and  swojlen. 

Highly     refractive, 
comparable     to     waxy 
tube-cast. 

Distinct. 

Sluggish. 

Smaller. 

Much  coarser,  darker 
in  color,  peripherally  ar- 
ranged.   Motion  slight. 

Usually    typical 
"daisy."     6  to  12  seg- 
ments. 

Same  as  tertian. 

Generally  darker  than 
normal. 

Highly  refractive. 

Distinct. 
Active. 

Young  forms,   only, 
in  peripheral  blood. 

Very    few,     minute, 
inactive.         Distinctly 
pigmented    forms   sel- 
dom seen. 

Very  rarely  seen  in 
peripheral  blood. 

Appear  in  blood  as 
distinctive  ovoids  and 
crescents. 

Dark,  often  bronzed. 

BLOOD   PARASITES  357 

one  should,  while  searching,  keep  a  sharp  lookout  for 
unusually  large  or  pale  corpuscles,  and  for  anything 
which  is  brown  or  black  or  in  motion. 

The  preceding  table  contrasts  the  distinguishing  char- 
acteristics of  the  three  varieties  as  seen  in  fresh  blood. 

(b)  In  Stained  Films  (See  Frontispiece  and  Plate 
VII) . — Recognition  of  the  parasite,  especially  the  young 
forms,  is  much  easier  in  films  stained  by  Wright's  or 
Some  similar  stain  than  in  fresh  blood.  The  films  must 
be  thin  and  well  stained.  It  is  useless  to  search  prepar- 
ations in  which  the  nuclei  of  leukocytes  are  not 
strongly  colored. 

In  films  which  ajre  properly  stained  with  Wright's  or 
Giemsa's  stain  malarial  parasites  appear  as  follows: 

The  young  parasites  are  small,  round,  ring-like  or 
irregular,  sky-blue  bodies,  each  with  a  very  small, 
sharply  defined,  purplish  red  chromatin  mass.  Many 
structures — deposits  of  stain,  dirt,  blood-platelets  lying 
upon  red  cells  (see  Fig.  134),  etc. — may  simulate  them, 
but  should  not  deceive  one  who  looks  carefully  for  both 
the  blue  cytoplasm  and  the  purplish  red  chromatin.  A 
platelet  upon  a  red  corpuscle  is  surrounded  by  a  color- 
less zone  rather  than  by  a  distinct  blue  body  and  there 
is  no  compact  chromatin  mass.  As  quartan  parasites 
grow  a  little  older  they  tend  to  assume  a  slender,  straight 
or  slightly  curved,  band-like  foim  which  is  fairly 
characteristic  of  this  species.  Young  estivo-autum- 
nal  parasites  commonly  take  the  form  of  small,  delicate, 
blue  rings,  each  with  one  or  two  small  purplish  red 
chromatin  bodies  upon  its  circumference.  Their  recog- 
nition is  important  because  they  may  be  the  only  form 
found  in  a  given  case.     When  young  tertian  and  quar- 


358  THE  BLOOD 

tan  parasites  assume  this  form  the  ring  is  usually  larger 
and  thicker.  Usually  it  is  the  dot-like  chromatin  body 
which  first  attracts  one's  attention  to -the  parasitized 
cell.  In  tertian  malaria  the  fact  that  cells  which  harbor 
the  parasites  are  somewhat  larger  and  paler  than  their 
fellows  is  also  helpful  in  attracting  one's  attention  while 
searching.  This  may  be  evident  as  early  as  eight  hours 
after  the  chill.  No  such  enlargement  of  the  red  cells 
is  noted  in  other  forms  of  malaria. 

Older  tertian  and  quartan  parasites  show  larger 
sky-blue  bodies  with  more  abundant,  paler,  and  more 
reticular  or  granular  chromatin,  and  contain  brown 
granules  of  pigment,  which,  however,  are  less  evident 
than  in  the  living  parasite.  The  chromatin  usually 
lies  in  a  colorless  area  or  "achromatic  zone"  and  is 
sometimes  so  pale  as  to  be  difficult  to  see  clearly.  Not 
infrequently  it  appears  to  lie  entirely  outside  of  the 
cytoplasm.  The  pigment  of  the  adult  tertian  parasite 
is  usually  fine  and  scattered  uniformly  through  the 
cytoplasm.  That  of  the  quartan  is  coarser  and  more 
peripherally  arranged.  The  corresponding  stage  of  the 
estivo-autumnal  parasite  rarely  appears  in  the  blood. 

Typical  "segmenters"  present  a  ring  of  rounded 
segments  or  spores,  each  with  a  small,  dot-like  chroma- 
tin mass,  but  these  regular  forms  are  not  often  seen. 
With  the  tertian  parasite,  especially,  the  segments 
much  more  frequently  form  an  irregular  cluster.  The 
pigment  is  collected  near  the  center  or  at  one  side  or  is 
scattered  among  the  segments. 

Fully  grown  tertian  and  quartan  gametes  resemble 
the  fully  grown  asexual  forms  in  general  appearance, 
but  are  more  compact  and  less  irregular  in  shape  and 


PLATE  VII 


Malarial  parasites.    Wright's  stain.      X  looo  (i  mm.  =  i  micron). 


e 


m5k 


• 


« 


(^ 


Fig.  I. — Estivo-autumnal  malaria,  exact  reproduction  of  a  portion  of  a  field. 


FiK.     2. — Estivo-autumnal 
-  gametes. 


i 


^^^ 

s 


I'lj;.    3. — Leukocytes    with 
engulfed  pigment. 


Fig.  4. — Quartan  parasites. 


Q- 


i^     *^-..'i 


B 


-V'^v 


^^*  c 


•V7/* 


Fig.  s.— Tertian  parasites:  A,  Eight  hours  after  chill,  showing  malarial  stippling,  five 
young  parasites,  and  one  gamete;  from  two  slides;  B,  twenty-four  hours  after  chill,  five 
half-grown  parasites,  one  gamete;  C,  during  chill,  one  presegmenter,  two  segmenters,  a 
cluster  of  freshly  liberated  merozoites,  and  two  very  young  parasites;  from  one  slide. 

(J.  W.  Rennell,  pinx.) 


BLOOD    PARASITES  359 

contain  more  and  larger  pigment  granules.  The  female 
is  generally  the  larger  and  has  more  compact  chromatin 
and  deeper  blue  cytoplasm.  The  crescentic  and  oval 
gametes  of  estivo-autumnal  malaria  are  easily  identified. 
Their  length  is  somewhat  greater  than  the  diameter  of 
a  red  corpuscle.  Their  chromatin  is  usually  centrally 
placed,  and  they  contain  more  or  less  coarse  pigment. 
The  remains  of  the  red  cell  often  form  a  narrow  rim 
around  them  or  fill  the  concavity  of  the  crescent. 

Concentration  Methods  for  Malarial  Parasites.— 
When  parasites  are  scarce  they  may  sometimes  be 
found,  although  their  structure  is  not  well  shown,  by 
the  Ross-Ruge  thick-smear  method.  This  consists  in 
spreading  a  very  thick  layer  of  blood,  drying,  placing 
for  a  few  minutes  in  a  fluid  containing  5  per  cent,  forma- 
lin and  I  per  cent,  acetic  acid,  which  removes  the  hemo- 
globin and  fixes  the  smear,  rinsing,  drying,  and  finally 
staining.  Carbol-thionin  is  very  useful  for  this  pur- 
pose. If  Wright's  stain  be  used  it  is  recommended 
that  the  preparation  be  subsequently  stained  for  a  half 
minute  with  borax-methylene-blue  (borax,  5 ;  methylene 
blue,  2;  water,  100).  Estivo-autumnal  crescents  may 
also  be  concentrated  by  the  method  given  for  filarial 
larvae  (p.  363).  These  older  methods  are,  however,  far 
inferior  to  the  following  new  method  of  Bass  and  Johns, 
which  takes  advantage  of  the  fact  that  parasitized  red 
cells  are  lighter  than  the  others  and  rise  to  the  top  of 
the  sediment  when  the  blood  is  centrifugalized  at  high 
speed.  A  centrifuge  capable  of  2500  revolutions  per 
minute  is  required. 

I.  Draw  10  c.c.  of  blood  from  a  vein  (see  p.  254) 
directly  into  a  tube  containing  0.2  c.c.  of  citrate-dextrose 


36o 


THE  BLOOD 


solution.  Mix  well.  The  solution  is  made  by  dissolving 
50  Gm.  sodium  citrate  and  50  Gm.  dextrose  in  100  c.c. 
distilled  water  by  the  aid  of  heat. 

2.  Divide  the  blood  between  two  centrifuge  tubes  and 
centrifugate  at  2500  revolutions  per  minute  for  the  proper 
length  of  time,  which  is  determined  by  the  radius  of  the 


A  B 

Fig.  138. — Estivo-autumnal  malaria:  effect  of  concentration  by- 
Bass  and  Johns's  method.  A,  direct  smear,  averaging  one  crescent  in 
eight  fields;  B,  blood  of  same  patient  concentrated.  From  slides  pre- 
pared by  F.  M.  Johns.     Wright's  stain  (  X  looo). 

centrifuge  arm  and  the  height  of  the  column  of  blood  in 
the  tube.  For  a  centrifuge  whose  radius  is  18  cm.  the 
proper  time  is  one  minute  for  each  centimeter  of  the  blood 
column.  Too  long  centrifugation  will  cause  the  corpuscles 
to  pack  so  tightly  that  the  subsequent  skimming  is  difficult; 
too  little  centrifugation  will  fail  to  bring  the  parasites 
to  the  top. 


BLOOD   PARASITES  36 1 

3.  The  leukocytes  and  all  malarial  parasites  (except 
very  young  forms)  will  now  be  concentrated  in  a  layer  i  mm. 
thick  at  the  top  of  the  sediment.  With  a  capillary  pipet 
(Fig.  228)  skim  off  this  layer  and  place  it,  together  with  a 
like  amount  of  plasma,  in  a  tube  about  12  cm.  in  length  and 
0.5  cm.  in  inside  diameter.  If  the  column  of  fluid  exceeds 
5  cm.,  two  tubes  should  be  used.  These  tubes  are  readily 
made  from  ordinary  glass  tubing. 

4.  Mix  thoroughly  and  centrifugate  as  before. 

5.  With  a  large  capillary  pipet  skim  off  the  top  layer 
of  the  sediment  in  these  tubes,  taking  up  a  column  of  cells 
and  plasma  not  exceeding  5  cm.  in  height. 

6.  Mix  by  forcing  in  and  out  upon  a  slide,  and  then  draw 
the  mixture  into  the  pipet  away  from  the  tip  and  seal  the 
tip  in  a  flame.  Nick  with  a  file  and  break  off  the  capillary 
stem  above  the  blood  column. 

7.  Place  this  slender  tube  in  the  centrifuge  and  revolve 
as  before.  The  leukocytes  will  form  a  grayish  layer  upon 
the  surface  of  the  sediment.  This  and  the  upper  portion 
of  the  erythrocyte-layer  contains  the  parasites. 

8.  Nick  with  a  file  and  break  off  the  capillary  tube  at 
a  point  I  to  2  mm.  below  the  bottom  of  the  leukocyte 
layer. 

9.  With  a  capillary  pipet  whose  stem  will  pass  inside 
the  capillary  tube  remove  the  small  amount  of  red  cells  and 
leukocytes  together  with  a  little  plasma. 

10.  Mix-  well,  make  smears  on  slides  and  stain  with 
Wright's  stain  in  the  usual  way. 

The  authors  of  this  method  claim  that  90  per  cent, 
of  the  parasites  in  10  c.c.  of  blood  can  be  collected  upon 
one  slide.  While  it  is  not  to  be  expected  that  such 
remarkable  concentration  can  be  attained  without  con- 
siderable experience  yet  the  method  will  yield  good  re- 
sults at  the  first  trial  if  the  directions  are  carefully 


362 


THE  BLOOD 


followed.  Best  results  are  obtained  with  estivo-autum- 
nal  crescents  and  adult  tertian  and  quartan  parasites. 
The  very  young  parasites  do  not  concentrate  well,  if 
at  all.  A  decided  advantage  over  the  other  methods  is 
the  fact  that  parasites  and  all  blood  cells  are  perfectly 
preserved  and  stain  exactly  as  they  do  in  ordinary 
smears  (see  Fig.  138  and  Plate  VI). 

4.  Filarial  Larvae.— A  description  of  the  filariae 
whose  larvre  appear  in  the  blood  will  be  found  on  page 
498.     Owing   to   the  remarkable  periodicity  of  their 


Fig.   139. — Fila.r:j.'.  l^^rva;  ir.  bl-'-c.      i-._.:.-_.      Red  corpuscles  decol- 
orized; a  few  leukocytes  remain  (  X  200). 


appearance  in  the  peripheral  circulation  the  most 
favorable  time  of  day  for  the  examination  for  micro- 
filariae will  depend  upon  the  species. 

When  numerous,  they  are  easily  found  in  fresh  un- 
stained blood.  A  rather  large  drop  is  taken  upon  a 
slide,  covered,  and  examined  with  a  low  power.  They 
can  be  located  by  the  commotion  which  their  active 
motion  produces  among  the  corpuscles.  This  motion 
consists  almost  wholly  in  apparently  purposeless  lash- 


BLOOD   P.\RASITES  363 

ing  and  coiling  movements,  and  continues  for  man\- 
hours  or  even  days  if  the  preparation  be  ringed  with 
vaselin  and  kept  in  a  cool  place.  If  desired,  stained 
smears  of  the  blood  may  be  prepared  in  the  usual  way 
or  by  the  Ross-Ruge  method  (p.  359).  When  the 
micro-filariae  are  scarce  the  following  method  is  efficient : 

Receive  about  i  c.c.  of  blood  from  a  puncture  of  the 
ear  or  finger  into  5  c.c.  of  2  per  cent,  acetic  acid.  Mix 
well  and  centrifugalize.  Spread  the  sediment,  which 
is  not  abundant,  upon  slides  and  examine  in  the  moist 
state  or  after  drying,  fixing  and  staining.  Hema- 
toxylin is  a  good  stain  for  the  purpose. 

The  number  of  micro-filariae  in  capillary  blood  is 
said  to  be  distinctly  higher  than  in  that  obtained  from 
a  vein. 

5.  Larvae  of  Trichinella  spiralis. — The  worm  and 
its  life  history  are  described  on  page  508.  In  1909 
Herrick  and  Janeway  demonstrated  that  diagnosis  of 
trichiniasis  can  frequently  be  made  by  detection  of  the 
larvfe  in  the  blood  during  their  migration  to  the  muscles. 
Of  the  examinations  which  have  been  reported  since 
that  time,  about  one-half  have  been  positive.  The 
earliest  time  at  which  the  embryos  were  found  was  the 
sixth  day  after  the  onset  of  symptoms;  the  latest,  the 
twenty-second  day.         • 

The  approved  method  is  the  same  as  that  given  above 
for  micro-filariae  except  that  5  or  10  c.c.  of  blood  from  a 
vein  (see  p.  254)  and  a  correspondingly  larger  quantity 
of  acetic  acid  solution  are  required.  The  larvae  are 
not  difficult  to  recognize.  They  are  about  125  ^  long 
and  6  n  broad. 


364  THE  BLOOD 

XL  TESTS  FOR  RECOGNITION  OF  BLOOD 

The  recognition  of  red  blood-corpuscles  microscopic- 
ally is  the  surest  and  simplest  means  of  detecting  the 
presence  of  blood.  In  most  pathologic  material,  how- 
ever, the  corpuscles  are  too  much  disintegrated  for 
recognition  with  the  microscope,  and  one  has  to  rely 
upon  a  test  for  hemoglobin  or  its  derivatives.  Of  such 
tests,  those  given  in  this  section  are  probably  the  best. 
Each  is  reliable  within  its  own  sphere,  but  each  has  its 
limitations.  The  guaiac,  benzidin  and  similar  tests  are 
reliable  only  when  negative.  When,  however,  proper 
care  is  taken  to  exclude  fallacies,  they  are  the  most  use- 
ful and  reliable  tests  for  clinical  purposes,  although  they 
could  not  be  accepted  medico-legally.  The  hemin  test 
is  reliable  only  when  positive.  The  spectroscope  offers 
perhaps  the  most  simple  and  dependable  means  of 
identifying  blood,  but,  except  under  favorable  condi- 
tions, it  is  not  adapted  to  the  detection  of  traces.  Its 
particular  field  lies  in  distinguishing  between  the  vari- 
ous hemoglobin  derivatives. 

The  only  reliable  test  for  human  blood  as  distinguished 
from  that  of  animals  is  the  precipitin  test  described  on 
page  611. 

1.  Guaiac  Test. — The  technic  of  this  test  has  been 
given  (see  p.  181).  It  may  be  applied  directly  to  a  sus- 
pected fluid,  but  in  order  to  avoid  other  substances 
which  might  cause  the  reaction  the  following  procedure 
is  advised:  Remove  fat  if  present  (e.g.,  in  feces)  by 
shaking  with  an  equal  volume  of  ether  and  discarding 
the  ether.  Add  3  or  4  c.c.  of  glacial  acetic  acid  to  about 
10  c.c.  of  the  fat-free  fluid;  shake  thoroughly  with  an 


TESTS    FOR   RECOGNITION    OF  BLOOD  365 

equal  volume  of  ether;  decant,  and  apply  the  test  to  the 
ether.  Should  the  ether  not  separate  well  add  a  little 
alcohol  and  mix  gently.  It  should  then  separate  nicely. 
Jager  states  that  the  test  is  rendered  much  more  sensi- 
tive if  a  few  drops  of  ammonia  or  sodium  hydroxid 
solution  be  added  to  the  ether  extract.  In  case  of  dried 
stains  upon  cloth,  wood,  etc.,  dissolve  the  stain  in  dis- 
tilled water  and  test  the  water,  or  press  a  piece  of  moist 
blotting-paper  against  the  stain,  and  touch  the  paper 
with  drops  of  the  guaiac  and  the  turpentine  successively. 
The  test  may  be  applied  to  microscopic  particles  by 
running  the  reagents  under  the  cover-glass. 

The  benzidin  test  (see  p.  182)  is  similar  to  the  guaiac 
test  and  has  the  same  fallacies,  but  is  distinctly  more 
sensitive. 

2.  Teichmann's  Test. — This  depends  upon  the  pro- 
duction of  characteristic  crystals  of  hemin.  It  is  not 
sufficiently  delicate  to  detect  the  minute  quantities  of 
blood  with  which  we  frequently  have  to  deal  in  the  clin- 
ical laboratory,  but,  when  positive,  it  is  absolute 
proof  of  the  presence  of  blood.  A  number  of  substances 
• — lime,  fine  sand,  iron  rust — interfere  with  production  of 
the  crystals;  hence  negative  results  are  not  always  con- 
clusive. Dissolve  the  suspected  stain  in  a  few  drops 
of  normal  salt  solution  upon  a  slide.  If  a  liquid  is  to  be 
tested,  evaporate  s'ome  of  it  upon  a  slide  and  dissolve 
the  residue  in  a  few  drops  of  the  salt  solution.  Let  dry, 
apply  a  cover-glass,  and  run  glacial  acetic  acid  under- 
neath it.  Heat  very  gently  until  bubbles  begin  to  form, 
replacing  the  acid  as  it  evaporates.  Allow  to  cool 
slowly.  When  cool,  replace  the  acid  with  water,  and 
examine  for  hemin  crystals  with  i6-mm.  and  4-mm. 


366 


THE  BLOOD 


objectives.  The  crystals  are  dark-brown  rhombic 
plates,  lying  singly  or  in  crosses,  and  easily  recognized 
(Fig.  140).     Failure  to  obtain  them  may  be  due  to  too 


Pig.   140. — Teichmann's  hemin  crystals  (Jakob). 

much  salt,  too  great  heat,  or  too  rapid  cooling.  If  not 
obtained  at  first,  let  the  slide  stand  in  a  warm  place,  as 
upon  a  hot-water  radiator,  for  an  hour. 


Pig.  141. — Small  direct-vision  spectroscope  with  ade  mirror.     About 
natural  size. 

3.  Spectroscopic  Method. — Spectrum  analysis  de- 
pends upon  the  fact  that  solutions  of  many  substances, 
when  held  so  as  to  intercept  the  light  entering  the 


TESTS    FOR   RECOGNITION    OF  BLOOD 


367 


o 

tu 
o: 


B   C 


UJ 

^ 

0 

0 

i: 

_j 

^ 

tu 

0 

>- 

z: 

LU 


D 


Eb 


I 


CQ 


- 

1 1 

■ 

II 

Solar  spectrum 
stiowinq  FraunhoFers 
lines 


Oxyhemoqlobln 

Hemoqlobin 
(Reduced  hemoqiobinj 

Methemoqlobin 


Hematin  in  acid 
so\wt\on 

Hematin  in  alkaline 
solution 

Reduced  alkali  hematin 

(Hemochromoqen) 

Hematoporph/nn 
in  acid  solution 

Carbon  monoxide 
hemoglobin. 


Fig.  142. — Absorption  spectra  of  hemoglobin  and  its  derivatives. 


368  THE  BLOOD 

spectroscope,  will  absorb  certain  colors,  thus  causing 
dark  bands  to  appear  at  definite  locations  in  the 
spectrum.  A  small  direct-vision  instrument  meets 
all  ordinary  requirements  and  may  be  recommended 
as  a  useful  addition  to  the  regular  laboratory  equip- 
ment. The  form  with  a  side  mirror  and  reflecting 
prism  (Fig.  141)  which  gives  two  spectra  side  by  side 
is  most  convenient.  Before  use,  the  width  of  the  sHt 
should  be  so  adjusted  and  the  eye-piece  so  focused  that 
Fraunhofer's  lines  (Fig.  142,  B,  C,  D,  E,  b,  F)  are  clearly 
seen,  since  it  is  by  means  of  these  lines  that  the  ab- 
sorption bands  are  located.  The  examination  is 
best  made  by  daylight.  With  artificial  light  the 
Fraunhofer  lines  do  not  appear.  The  solution  under 
examination  may  be  held  in  a  test-tube  or  small 
beaker.  If  a  test-tube  be  used,  only  i  to  3  c.c.  will  be 
required. 

The  treatment  of  the  suspected  material  will  depend 
upon  its  condition  and  the  purpose  of  the  examination: 

1.  When  fresh  blood  is  studied  for  oxyhemoglobin, 
methemoglobin,  etc.,  a  large  drop  from  a  skin  puncture  is 
received  in  i  or  2  c.c.  of  water  in  a  test-tube  and  cautiously 
diluted  to  the  point  where  the  bands  become  distinct.  The 
optimum  dilution  is  much  less  for  methemoglobin  than  for 
oxyhemoglobin. 

2.  Urine  and  other  fluids  suspected  to  contain  blood  may 
be  cleared  by  filtration  and  examined  directly.  When  this 
proves  unsatisfactory,  as  is  often  the  case  owing  to  per- 
sistent cloudiness  or  the  presence  of  other  pigments  which 
darken  the  whole  spectrum,  the  blood  pigment  in  200  to 
500  c.c.  of  the  unfiltered  fluid  should  be  extracted  as  fol- 
lows: Add  a  little  white  of  egg  if  the  fluid  is  not  already 
sufficiently  albuminous,  boil,  acidify,  centrifugalize,  remove 


TESTS  FOR  RECOGNITION  OF  BLOOD      369 

supernatant  fluid  and  treat  the  sediment  as  described  in 
the  following  paragraph. 

3.  Feces,  gastric  contents  and  other  material  should 
be  treated  with  glacial  acetic  acid  and  extracted  with  ether 
as  described  under  the  guaiac  test  (p.  364).  Blood-pigment 
is  thus  changed  to  acid  hematin,  which  is  taken  up  by  the 
acidified  ether,  giving  a  clear  solution  suitable  for  spectro- 
scopic examination.  If  the  ether  does  not  take  up  the 
blood-pigment  well,  a  little  more  acetic  acid  should  be 
added.  In  order  that  the  solution  may  not  be  too  dilute 
to  show  the  bands,  a  less  amount  of  ether  than  is  recom- 
mended for  the  guaiac  test  may  be  used,  or  the  ether-extract 
may  be  concentrated  by  evaporation. 

When  the  result  is  in  doubt  the  acid  hematin  may  be 
transformed  into  the  more  easily  identified  hemochromogen 
as  follows:  Render  the  ethereal  extract  alkaline  with  strong 
ammonia,  cooling  if  necessary,  mix  well,  and  let  stand  until 
the  fluids  separate.  The  ammonia  will  contain  alkali 
hematin.  By  means  of  a  pipet  transfer  it  to  another  test- 
tube  and  add  a  few  drops  of  fresh  ammonium  sulphid  or 
Stokes'  reagent.  The  bands  of  hemochromogen  should 
appear  at  once. 

4.  Stains  of  blood  dried  on  clothing,  etc.,  should  be 
dissolved  in  i  or  2  c.c.  of  10  per  cent,  caustic  soda  solution, 
heated  to  a  point  just  short  of  boiling,  cooled,  and  treated 
with  a  few  drops  of  ammonium  sulphid  or  Stoke's  reagent. 
The  solution  is  then  examined  for  the  characteristic  bands 
of  hemochromogen. 

5.  In  very  old  blood  stains  the  hemoglobin  may  have  been 
transformed  to  the  iron-free  pigment  hematoporphyrin, 
which  is  very  resistent  to  solution.  It  will  usually  dissolve 
in  strong  sulphuric  acid.  It  has  been  advised  to  place  a 
few  small  bits  of  the  dry  stain  on  a  slide  in  a  drop  of  con- 
centrated sulphuric  acid,  to  apply  a  cover  and  rub  the 
bits  of  blood  between  slide  and  cover.     Enough  may  go 

24 


370  THE   BLOOD 

into  solution  to  admit  of  spectroscopic  examination. 
Particles  of  wood,  cloth,  or  other  organic  material  which 
might  blacken  the  acid  should  be  avoided. 

The  characteristic  absorption  spectra  of  the  more 
important  hemoglobin  derivatives  are  as  follows: 

1.  Oxyhemoglobin  is  present  only  in  comparatively  fresh 
blood.  It  gives  two  dark  bands  between  the  lines  D  and  E. 
In  concentrated  solution  these  unite  to  form  a  single  broad 
band.  Upon  addition  of  a  few  drops  of  fresh  ammonium 
sulphid,  or,  much  better,  Stokes'  reagent,^  the  spectrum" 
changes  to  that  of  reduced  hemoglobin. 

2.  Hemoglobin  (also  called  reduced  hemoglobin)  gives 
a  single  broad  band  between  D  and  E.  By  shaking  with  air 
it  is  changed  to  oxyhemoglobin  whose  bands  in  the  same  di- 
lution are  more  distinct. 

3.  Methemoglobin  occurs  in  the  circulating  blood 
under  the  conditions  which  have  been  described  (see  p.  260). 
It  may  also  be  found  in  the  urine,  in  hemorrhagic  cyst 
fluids,  etc.  In  neutral  or  faintly  acid  solution,  its  most 
characteristic  band  is  situated- between  the  lines  C  and  D. 
Two  less  distinct  bands  lie  between  D  and  E  and  a  broad 
one  beyond  E;  but  these  are  usually  not  clearly  seen.  The 
blood  must  be  diluted  cautiously,  as  it  is  easy  to  pass  the 
point  where  the  characteristic  band  is  most  distinct. 
Upon  addition  of  a  few  drops  of  fresh  ammonium  sulphid 
or  Stokes'  reagent,  methemoglobin  is  changed  to  reduced 
hemoglobin  with  its  single  broad  band.  This  will  serve  to 
distinguish  it  from  acid  hematin. 

Methemoglobin  can  be  prepared  for  purposes  of  compar- 

^  Stokes'  reagent  consists  of  ferrous  sulphate,  2  Gm.;  tartaric  acid 
3  Gm.;  water,  100  c.c.  When  needed  for  use  take  a  few  cubic  centi- 
meters in  a  test-tube  and  add  strong  ammonia  drop  by  drop  until  the 
precipitate  which  forms  at  first  has  entirely  dissolved. 


TESTS    FOR    RECOGNITION    OF   BLOOD  37 1 

ison  by  diluting  2  drops  of  blood  with  20  drops  of  water, 
adding  i  or  2  drops  of  strong  potassium  ferricyanid  solution, 
and  shaking.  The  solution  turns  chocolate  brown,  and 
may  then  be  diluted  until  the  characteristic  band  is  distinct. 

4.  Hematin  may  be  formed  through  the  action  of  acids 
or  alkalies  as  in  gastric  and  intestinal  bleeding.  It  is 
sometimes  found  in  old  extravasates,  in  the  urine,  and 
elsewhere.  It  is  insoluble  in  water  or  weak  acids,  readily 
soluble  in  acidified  ether  and  weak  alkalies. 

As  seen  from  Fig.  142,  the  absorption  bands  of  hema- 
tin in  acid  solution  ("acid  hematin")  are  similar  to  those 
.of  methemoglobin.  That  between  C  and  D  is  most  def- 
inite; the  others  may  not  be  clearly  seen.  In  contrast 
to  methemoglobin  the  addition  of  ammonium  sulphid 
or  Stokes'  reagent  does  not  produce  the  spectrum  of  re- 
duced hemoglobin  but  rather  (if  the  solution  has  been 
sufficiently  alkalinized  to  produce  alkali  hematin)  that  of 
hemochromogen. 

Hematin  in  alkaline  solution  ("alkali  hematin")  gives  a 
rather  indefinite  broad  band  between  C  and  D.  Its 
presence  may  be  confirmed  by  adding  a  few  drops  of 
ammonium  sulphid  or  Stokes'  reagent.  The  solution 
becomes  brighter  red  in  color,  and  the  spectrum  changes  to 
the  more  easily  identified  one  of  hemochromogen. 

5.  Hemochromogen,  also  called  reduced  alkali  hematin, 
gives  a  narrow,  very  distinct  band  between  D  and  E,  and  if 
not  in  too  dilute  solution,  a  fainter  band  between  E  and  b. 
This  is  one  of  the  most  definite  and  characteristic  of  the 
blood-pigment  spectra. 

6.  Hematoporphyrin  is  an  iron-free  hemoglobin  deriva- 
tive which  may  occasionally  be  present  in  the  urine, 
especially  in  sulphonal  poisoning  (see  p.  184)  and  in  very 
old  dried  blood.  It  does  not  respond  to  the  guaiac  or 
hemin  test.  It  is  soluble  in  strong  sulphuric  acid.  Its 
absorption  spectrum  is  shown  in  Fig.  142. 


372  THE  BLOOD 

For  purposes  of  comparison  it  can  be  prepared  by  adding 
a  drop  of  blood  to  2  or  3  c.c.  of  concentrated  sulphuric 
acid. 

7.  Carbon  monoxid  hemoglobin,  which  appears  in  the 
circulating  blood  in  carbon  monoxid  poisoning,  gives  two 
bands  very  like  those  of  oxyhemoglobin,  but  somewhat 
nearer  the  violet  end  of  the  spectrum.  In  contrast  to 
oxyhemoglobin,  addition  of  ammonium  sulphid  or  Stokes' 
reagent  leaves  these  bands  unchanged.  Owing  to  the 
small  quantity  usually  present  in  poisoning  the  chemical 
test  is  preferable  for  its  detection  (see  page  261). 

XII.    LESS  FREQUENTLY  USED  METHODS 

In  this  section  brief  consideration  will  be  given  a 
number  of  methods  which  are  not  as  yet  in  common 
use,  some  because  their  clinical  value  has  not  been 
proved,  others  because  the  technic  has  not  been  suffi- 
ciently simplified. 

1.  Chemic  Examination. — In  routine  clinical  work 
chemic  study  of  the  blood  has  been  limited  to  estima- 
tions of  hemoglobin.  The  study  of  other  substances 
has  in  the  past  interested  the  biochemist  rather  than 
the  clinician.  Within  the  past  few  years,  however, 
methods  have  been  so  simplified  and  so  many  facts  of 
clinical  value  have  been  gathered  that  certain  chemic 
examinations  are  beginning  to  play  an  extremely  im- 
portant role  in  clinical  medicine.  Among  the  more 
useful  of  these  are  the  Lewis  and  Benedict  method  for 
blood  sugar;  the  picric  acid  method  for  creatinin;  and 
Folin's  new  direct  Nesslerization  methods  for  urea,  non- 
protein nitrogen  and  total  nitrogen.  All  of  these  are 
colorimetric  methods  and  detailed  directions  for  some 


LESS   FREQUENTLY   USED   METHODS  373 

of  them  are  given  in  the  printed  matter  which  accom- 
panies the  Hellige  and  the  Kuttner  colorimeters.^ 

2.  Vital  Staining. — Upon  the  assumption  that 
ordinary  staining  of  dried  and  fixed  blood-films  gives 
the  reactions  of  dead  cells  and  does  not  necessarily 
indicate  the  condition  of  the  living  blood,  attempts 
have  been  made  to  stain  blood  cells  in  the  living  state. 
The  information  yielded  by  this  so-called  "vital 
staining"  is  not  yet  of  much  value.  It  has  to  do 
chiefly  with  certain  "reticulated"  or  "skeined"  red 
corpuscles  which  contain  a  coarse  skein  or  network 
of  filaments  usually  confined  to  the  central  half  of  the 
cell.  The  filaments  stain  sharply  with  basic  dyes. 
Sometimes  discrete  granules  are  also  present.  Reticu- 
lation is  thought  to  be  a  characteristic  of  the  younger 
red  corpuscles.  Such  cells  constitute  about  0.3  to  i 
per  cent,  of  all  the  red  cells  in  the  blood  of  normal 
adults ;  an  increase  may  be  regarded  as  a  sign  of  active 
blood  regeneration.  They  are  more  abundant  in  child- 
hood. In  anemia,  particularly  in  pernicious  anemia, 
the  percentage  is  markedly  increased. 

The  following  method  has  proved  satisfactory  in 
the  writer's  laboratory: 

I.  In  a  small  test-tube  (about  10  X  75  mm.)  place  about 
3  drops  of  the  following  staining  solution  which  should  be 
freshly  mixed: 

Saturated  solution  brilliant  cresyl  blue  in  0.85  per 
cent,  salt  solution 7  c.c. ; 

Saturated  solution  of  neutral  potassium  oxalate .   2  c.c. 

1  These  and  other  similar  methods  have  recently  been  modified  for  use  with 
the  Denison  Laboratory  colorimeter  and  will  be  published  soon  by  R.  C.  Lewis 
and  A.  R.  Peebles. 


374  THE   BLOOD 

2.  Prick  the  ear  and  allow  one  drop  of  blood  to  fall 
into  the  stain. 

3.  Mix  gently  and  let  stand  ten  to  thirty  minutes.  A 
longer  time  will  do  no  harm. 

4.  Remove  small  drops  of  the  fluid,  make  smears  on 
slides  and  dry  in  the  air.  Examine  with  an  oil-immersion 
lens.     Preparations  begin  to  fade  after  a  day  or  two. 

In  such  preparations  the  leukocytes  and  platelets  are 
colored  shades  of  blue  and  the  red  corpuscles,  pale  greenish 
yellow.  The  skein  or  network  in  reticulated  cells  is  blue 
and  stands  out  distinctly  (see  Plate  V). 

3.  Resistance    of    the    Red    Corpuscles. — Many 

agencies  are  capable  of  causing  hemoglobin  to  pass 
from  the  red  corpuscles  into  the  surrounding  medium — 
a  phenomenon  which  is  known  as  hemolysis  and  which 
has  a  wide  interest.  Hemolysis  sometimes  occurs  in  the 
circulating  blood  and  may  then  be  due,  in  part  at  least, 
to  lowered  resistance  (or  "increased  fragility")  upon  the 
part  of  the  red  cells.  The  resistance  of  the  red  cells  can 
be  measured  by  subjecting  them  to  the  action  of  various 
agents.     For  clinical  work  salt  solution  is  generally  used. 

Method. — I.  Receive  i  or  2  c.c.  of  blood,  preferab-y 
from  a  vein,  directly  into  a  graduated  centrifuge  tube 
containing  about  2  c.c.  of  citrated  salt  solution  (sodium 
chloride,  0.9  Gm.;  sodium  citrate,  0.5  Gm.;  water, >ioo  c.c). 
Mix  gently. 

2.  Wash  the  corpuscles  twice  with  0.7  per  cent,  salt  so- 
lution by  centrifugalizing  and  pipeting  off  the  supernatant 
fluid,  the  last  time  leaving  a  volume  of  fluid  equal  to  the 
volume  of  corpuscles.     Mix  gently. 

3.  Arrange  a  series  of  11  small  test-tubes  and  place  in 
each  I  c.c.  of  sodium  chlorid  solution  varying  in  strength 


LESS    FREQUENTLY    USED    METHODS  375 

from  0.2  per  cent,  in  the  first  tube  to  0.7  per  cent,  in  the  last. 
Thus  each  tube  will  differ  from  the  next  by  0.05  per  cent. 

4.  To  each  tube  add  o.i  c.c.  of  the  suspension  of 
washed  corpuscles  and  mix  by  inverting  once  or  twice. 
Instead  of  using  washed  corpuscles  some  workers  simply 
add  I  drop  of  blood  from  a  skin  puncture  to  each  tube. 

5.  Let  stand  two  hours  at  room  temperature.  At  the 
end  of  that  time  the  corpuscles  will  have  settled  to  the 
bottom  and  hemolysis  may  be  recognized  by  the  color  of 
the  supernatant  fluid:  faintly  pink,  if  hemolysis  is  partial 
("initial  hemolysis");  red,  with  little  or  no  sediment,  if 
it  is  complete. 

With  normal  blood,  hemolysis  usually  begins  in  the  tube 
containing  0.45  per  cent,  salt  solution  and  is  complete  in 
that  containing  0.35  per  cent.  In  chronic  family  icterus 
Hill  found  the  figures  for  initial  and  complete  hemolysis 
to  be  0.60  and  0.40  respectively;  in  obstructive  jaundice, 
0.40  and  0.225.  In  secondary  and  pernicious  anemia  his 
figures  vary  only  slightly  from  the  normal,  hemolysis 
usually  beginning  somewhat  earlier  and  ending  somewhat 
later  than  it  does  in  normal  blood. 

4.  Matching  Bloods  for  Transfusion. — Untoward 
results  which  sometimes  follow  transfusion  of  blood  are 
now  known  to  be  due  in  many  instances  to  hemolysis 
or  agglutination  of  the  red  corpuscles  of  either  the 
donor's  or  recipient's  blood  or  both.  By  a  simple  test 
it  is  possible  to  ascertain  whether  the  blood  of  any 
individual  is  suitable  in  this  respect  for  transfusion 
into  the  veins  of  a  given  patient.  This  is  known  as 
"matching  bloods,"  and  if  possible  it  should  always  be 
done  when  transfusion  is  contemplated.  The  two 
factors  to  be  considered  are  hemolysis  and  agglutina- 
tion, but,  since  hemolysis  does  not  occur  without  agglu- 


376  THE   BLOOD 

tination,  it  is  suificient  in  practice  to  test  for  agglutina- 
tion only.  It  is  an  interesting  fact  that  in  respect  to 
the  presence  or  absence  of  iso-agglutinin  every  adult 
falls  into  one  of  four  groups  which  for  convenience  are 
designated  I,  II,  III,  and  IV.  To  explain  this  grouping 
it  is  necessary  to  assume  the  existence  of  two  distinct 
agglutinins  which  may  be  present  alone  or  in  combina- 
tion or  which  may  both  be  absent.  A  given  blood  will 
agglutinate  (and  often  hemolyze)  the  red  corpuscles  of 

No  agglutinin.  /0%  ofadults.  One  agiffiuiininjB"  7J^ of  adults. 

©C ~:::p® 


One  a^kjtinin'A','40%ofcK/u/is.       Two  a^ff&ifthins,A<iJB"^3%ofa<Ale3. 


Fig.  143. — Diagram  showing  the  interrelation  of  the  four  iso- 
agglutination  groups  of  Moss.  The  serum  of  any  group  will  aggluti- 
nate the  corpuscles  of  those  groups  toward  which  its  arrows  point. 
Thus,  serum  of  an  individual  belonging  to  Group  IV  will  agglutinate 
red  corpuscles  belonging  to  any  other  group,  while  the  serum  of 
Group  I  lacks  agglutinating  power  (after  Sanford). 

any  blood  which  lacks  agglutinin  of  the  same  kind  and 
will  have  no  effect  upon  blood  which  contains  the  same 
agglutinin.  There  can  therefore  be  neither  agglutina- 
tion nor  hemolysis  between  members  of  the  same 
group.  The  four  groups  and  their  interrelationship  is 
well  shown  in  Fig.  143.  It  is  thought  that  the  group 
to  which  an  individual  belongs  is  an  inherited  char- 
acteristic which  follows  Mendel's  law,  although  it  does 
not  become  fixed  until  after  the  period  of  infancy. 


LESS    FREQUENTLY   USED    METHODS  377 

When  transfusion  is  undertaken,  the  blood  should  be 
secured  from  an  individual  belonging  to  the  same  group 
as  the  patient.  If  such  a  donor  can  not  be  found,  as 
may  easily  happen  if  the  patient  belongs  to  either  of 
the  small  groups,  I  or  III,  blood  belonging  to  another 
group  may  be  used  provided  that  the  serum  of  the  patient 
does  not  agglutinate  the  corpuscles  of  the  donor. 

Moss'  Method  {Minot's  modification). — i.  Obtain  the 
following  from  each  of  the  two  persons  whose  blood  is  to  be 
matched: 

(a)  Red  cell  suspension.  Puncture  finger  or  ear  and  let 
a  large  drop  of  blood  fall  directly  into  a  small  test-tube 
containing  i  c.c.  of  a  1.5  per  cent,  solution  of  sodium  citrate 
in  0.9  per  cent,  salt  solution.  Mix  gently  by  inverting  a 
few  times. 

(b)  Serum.  Obtain  a  few  drops  of  blood  in  a  small  tube 
or  Wright  capsule  (see  Figs.  230  and  231).  As  soon  as 
coagulation  has  taken  place,  gently  loosen  the  clot  from  the 
wall  of  the  tube.  Let  stand  until  serum  has  separated  well. 
Separation  of  serum  can  be  hastened  by  centrifugation. 

2.  Make  thick  vaselin  rings  on  two  slides.  In  one  mix 
I  drop  each  of  the  patient's  serum  and  the  suspension  of 
the  donor's  corpuscles;  in  the  other  mix  i  drop  each  of  the 
patient's  corpuscles  and  the  donor's  serum.  The  fluids 
may  be  transferred  to  the  slide  by  means  of  a  capillary 
pipet  (see  Fig.  228)  or  a  platinum  loop.  Cover  each  of  the 
preparations  with  a  cover-glass,  and  at  the  end  of  about 
five  and  ten  minutes  re-mix  corpuscles  and  serum  by  lifting 
one  edge  of  the  cover. 

If  hollow-ground  slides  are  at  hand  hanging-drop  prepa- 
rations are  preferable.  The  corpuscles  and  serum  are  then 
mixed  at  intervals  by  tilting  the  slide. 

3.  At  intervals  examine  for  agglutination  of  red  corpus- 
cles with  a  low-power  objective.     When  agglutination  takes 


37^  THE   BLOOD 

place  the  corpuscles  gather  into  dense  irregular  clumps  or 
large  masses  (Fig,  144).  These  are  often  so  large  as  to  be 
seen  with  the  unaided  eye  as  fine  brick-red  granules. 
Clumping  is  usually  well  marked  within  a  few  minutes  but 
it  is  safe  to  allow  half  an  hour. 

The  only  important  source  of  error  is  rouleau  formation, 
which  may  or  may  not  occur  and  which,  although  the  clumps 


1 


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Fig.  144. — Matching  bloods  for  transfusion.  A,  corpuscles  of  a 
patient  with  serum  of  a  prospective  donor;  no  agglutination.  B, 
serum  of  patient  with  corpuscles  of  prospective  donor;  strong  aggluti- 
nation. The  blood  of  the  donor  is  therefore  unsuited  for  use  in  this 
case  (  X  100). 

are  usually  very  small,  might  not  be  easy  to  differentiate 
without  close  observation  with  the  4  mm.  objective.  In 
the  case  of  rouleau  formation  the  corpuscles  can  be  seen 
to  lie  in  rows  within  the  groups.  Re-mixing  of  the  cells 
and  serum  as  above  directed  tends  to  break  up  rouleaux 
and  to  favor  agglutination.     If  one  feels  uncertain  of  one's 


LESS    FREQUENTLY    USED    METHODS  379 

* 

interpretation,  one  should  make  control  slides  with  the 
patient's  serum  and  corpuscles  and  the  donor's  serum  and 
corpuscles.  Under  these  conditions  agglutination  will  not 
occur. 

To  Determine  the  Group  to  which  an  Individual  Belongs. 
—As  has  been  indicated  above  it  is  sufficient  in  a  given  case 
to  test  the  blood  of  a  series  of  prospective  donors  until 
one  is  found  which  matches  the  patient's  blood.  In  hos- 
pital work,  upon  the  other  hand,  it  will  be  found  much 
more  satisfactory  to  determine  in  advance  the  grouping 
of  a  number  of  individuals  who  may  be  willing  to  serve 
as  donors  upon  occasion.  When  an  emergency  arises, 
it  is  then  only  necessary  to  find  the  group  to  which  the 
patient  belongs  in  order  to  know  at  once  the  appropriate 
donor,  a  procedure  which  does  not  require  more  than 
half  an  hour. 

The  group  to  which  any  individual  belongs  is  easily 
ascertained  by  testing  his  serum  and  corpuscles  against  the 
corpuscles  and  serum  of  an  individual  known  to  belong  to 
Group  II  or  III,  using  the  simple  method  described  above. 
Interpretation  of  results  is  made  clear  by  Fig.  143.  If,  for 
example,  the  unknown  blood  agglutinates  Group  II  blood 
and  is  not  agglutinated  by  it,  then  the  unknown  must  belong 
to  Group  IV.  The  same  end  may  be  accomplished  by 
testing  the  corpuscles  of  the  unknown  against  sera  of  both 
Groups  II  and  III.  Such  sera,  if  kept  sterile,  will  remain 
active  for  months  and  may  be  kept  on  hand  in  small  glass 
capsules  (Fig.  231)  the  ends  of  which  are  to  be  sealed  in 
the  flame. 

5.  Viscosity. — It  is  evident  that  variations  in  the 
viscosity  of  the  blood  must  markedly  influence  the  load 
carried  by  the  heart,  but  viscosity  estimations  have 
proved  of  comparatively  little  value.  The  greatest  field 
would  seem  to  be  in  suggesting  need  for  treatment  when 


380  THE  BLOOD 

• 

high  viscosity  is  throwing  an  excessive  burden  upon  an 
already  weakened  heart. 

Compared  with  distilled  water,  the  normal  viscosity 
is  about  4.5.  It  is  reduced  in  primary  and  secondary 
anemia  (roughly  proportional  to  the  grade  of  anemia), 
nephritis,  cardiac  lesions  with  edema,  and  usually  in 
leukemia  and  malaria.  It  is  increased  in  polycythemia, 
diabetes  mellitus,  icterus,  and  usually  in  pneumonia. 
Measurement  of  viscosity  is  comparatively  simple  if  one 
has  a  suitable  instrument.  The  Hess  instrument  is  one 
of  the  best  and  is  accompanied  by  directions  for  use. 

XIII.    SPECIAL  BLOOD  PATHOLOGY 

The  more  conspicuous  characteristics  of  the  blood  in 
various  diseases  have  been  mentioned  in  previous  sec- 
tions. Although  the  great  majority  of  blood  changes 
are  secondary,  there  are  a  few  blood  conditions  in 
which  the  changes  are  so  prominent,  or  the  etiology 
so  obscure,  that  they  are  commonly  spoken  of  as  blood 
diseases.  These  will  receive  brief  consideration  here. 
They  fall  into  two  general  groups.  In  the  one  group 
(Anemia)  the  red  cells  and  hemoglobin  are  chiefly 
affected;  in  the  other  (Leukemia)  changes  in  the  leuko- 
cytes constitute  the  conspicuous  feature  of  the 
blood-picture. 

A.    Anemia 

This  is  a  deficiency  of  hemoglobin  or  red  corpuscles, 
or  both.  It  is  either  primary  or  secondary.  The  dis- 
tinction is  based  chiefly  upon  etiology,  although  each 
type  presents  a  more  or  less  distinctive  blood-picture. 
Secondary  anemia  is  that  which  is  symptomatic  of  some 


SPECIAL  BLOOD    PATHOLOGY  381 

other  pathologic  condition.  Primary  anemia  is  that 
which  progresses  without  apparent  cause.  While 
such  a  classification  is  unsatisfactory  from  a  scientific 
standpoint,  it  has  long  been  current  and  is  very  useful 
in  practice. 

1.  Secondary  Anemia. — The  more  important  con- 
ditions which  produce  secondary  or  symptomatic  ane- 
mia are: 

(a)  Poor  nutrition,  which  usually  accompanies  un- 
sanitary conditions,  poor  and  insufiicient  food,  etc. 

(b)  Acute  infectious  diseases,  especially  rheumatism 
and  typhoid  fever.  The  anemia  is  more  conspicuous 
during  convalescence. 

(c)  Chronic  Infectious  Diseases. — Tuberculosis,  syph- 
ilis, leprosy. 

{d)  Chronic  exhausting  diseases,  as  heart  disease, 
chronic  nephritis,  cirrhosis  of  the  liver,  and  gastro- 
intestinal diseases,  especially  when  associated  with 
atrophy  of  gastric  and  duodenal  glands.  The  last  may 
give  an  extreme  anemia,  indistinguishable  from  perni- 
cious anemia. 

(e)  Chronic  poisoning,  as  from  lead,  arsenic,  and 
phosphorus. 

(/)  Hemorrhage. — Either  repeated  small  hemorrhages 
(chronic  hemorrhage) ,  as  from  gastric  cancer  and  ulcer, 
hemorrhoids,  uterine  fibroids,  etc.,  or  acute  hemorrhage, 
such  as  may  occur  in  typhoid  fever,  tuberculosis,  or 
traumatism. 

(g)  Malignant  Tumors. — These  affect  the  blood  partly 
through  repeated  small  hemorrhages,  partly  through 
toxic  products,  and  partly  through  interference  with 
nutrition. 


382  THE  BLOOD 

{K)  Animal  Parasites. — Some  cause  no  appreciable 
change  in  the  blood;  others,  like  the  hookworm  and 
Dibothriocephalus  latus,  may  produce  a  very  severe 
anemia,  almost  identical  with  pernicious  anemia.  Ane- 
mia in  these  cases  is  probably  due  both  to  toxins  and 
to  abstraction  of  blood.  In  malaria  the  parasites  them- 
selves directly  destroy  the  red  cells. 

The  blood-picture  varies  with  the  grade  of  anemia. 
Diminution  of  hemoglobin  is  the  most  characteristic 
feature.  In  mild  cases  it  is  slight,  and  is  the  only  blood 
change  to  be  noted.  In  very  severe  cases  hemoglobin 
may  fa.l  to  15  per  cent,  or  even  lower.  Red  corpuscles 
are  diminished  in  all  but  very  mild  cases,  while  in  the 
severest  cases  the  red-corpuscle  count  is  sometimes  be^ 
low  1,000,000.     The  color-index  is  usually  decreased. 

Although  the  number  of  leukocytes  bears  no  relation 
to  the  anemia,  leukocytosis  is  common,  being  due  to  the 
same  cause. 

Stained  films  show  no  changes  in  very  mild  cases.  In 
moderate  cases  variations  in  size  and  shape  of  the  red 
cells  and  polychromatophilia  occur.  Very  severe  cases 
show  the  same  changes  to  greater  degree,  with  addition 
of  basophilic  degeneration  and  the  presence  of  normo- 
blasts in  small  or  moderate  numbers.  Megaloblasts  in 
very  small  numbers  have  been  encountered  in  certain 
severe  cases.  They  are  especially  abundant  and  may 
even  predominate  over  the  normoblasts  in  dibothrio- 
cephalus infection  and  in  the  anemia  of  malignant  dis- 
ease when  there  are  metastases  in  the  bone-marrow. 
Blood-platelets  are  usually  increased. 

Posthemorrhagic  Anemia. — Within  a  few  hours  after 
an  acute  hemorrhage  the  volume  of  blood  is  nearly  or 


SPECIAL  BLOOD   PATHOLOGY  383 

quite  restored  by  means  of  fluids  from  the  tissues. 
Owing  to  the  fact  that  some  destruction  of  red  corpus- 
cles continues  for  a  time,  the  anemia  is  most  marked 
a  few  days  after  the  hemorrhage.  Hemoglobin  and  red 
cells  are  diminished  according  to  the  amount  of  blood 
lost.  The  color-index  is  moderately  low.  There  is 
moderate  leukocytosis.  Some  of  the  red  cells  may 
show  polychromatophilia  and  a  few  normoblasts  may 
be  found.  In  some  cases  great  numbers  of  normoblasts 
appear  rather  suddenly — a  so-called  blood  crisis.  Nor- 
mal conditions  may  be  restored  within  a  few  weeks, 
although  the  color-index  is  apt  to  remain  low  for  some 
time  thereafter. 

2.  Primary  Anemia. — The  commonly  described 
varieties  of  primary  anemia  are  pernicious  anemia, 
aplastic  anemia  and  chlorosis,  but  splenic  anemia  may 
also  be  mentioned  under  this  head. 

( I )  Progressive  Pernicious  Anemia. — It  is  frequently 
impossible  to  diagnose  this  disease  from  the  blood  ex- 
amination alone.  Severe  secondary  anemia,  especially 
that  due  to  gastro-intestinal  cancer,  intestinal  parasites, 
and  repeated  small  hemorrhages,  sometimes  gives  an 
identical  picture.  Remissions,  in  which  the  blood  ap- 
proaches the  normal,  are  common.  All  the  clinical 
data  must,  therefore,  be  considered,  together  with  esti- 
mations of  urobilin  in  the  feces  (see  p.  430)  and  a 
careful  analysis  of  repeated  blood  examinations. 

The  disease  is  characterized  by  active  destruction  of 
red  corpuscles  with  excessive  activity  of  the  erythro- 
blastic bone-marrow,  and  the  appearance  of  immature 
and  abnormal  red  cells  in  the  circulation. 

Hemoglobin  and  red  corpuscles  are  always  greatly 


384  THE  BLOOD 

diminished.  Several  counts  in  which  the  red  cells 
were  below  150,000  have  been  recorded.  In  none  of 
Cabot's  139  cases  did  the  count  exceed  2,500,000,  the 
average  being  about  1,200,000.  In  more  than  two- 
thirds  of  the  cases  hemoglobin  was  reduced  to  less 
extent  than  the  red  corpuscles;  the  color-index  was, 
therefore,  high.  A  low  color-index  probably  indicates 
a  mild  type  of  disease.  The  average  hemoglobin 
value  is  about  20  to  25  per  cent. 

The  leukocyte  count  may  be  normal,  but  is  commonly 
diminished  to  about  3000,  and  is  sometimes  much  lower. 
The  decrease  affects  chiefly  the  polymorphonuclear 
cells,  so  that  the  lymphocytes  are  relatively  increased. 
In  some  cases  a  decided  absolute  increase  of  lympho- 
cytes occurs.  Polymorphonuclear  leukocytosis,  when 
present,  is  due  to  some  complication. 

The  red  corpuscles  show  marked  variation  in  size  and 
shape  (Plate  VIII  and  Fig.  145).  There  is  a  decided 
tendency  to  large  oval  forms,  and,  despite  the  presence 
of  microcytes,  the  average  size  of  the  corpuscles  is  gen- 
erally strikingly  increased.  Polychromatophilia  and 
basophilic  degeneration  are  common.  Nucleated  red 
cells  are  always  present,  although  in  many  instances 
careful  search  is  required  to  find  tliem.  In  the  great 
majority  of  cases  megaloblasts  exceed  normoblasts  in 
number.  This  ratio  is  practically  unknown  in  other 
diseases.     Blood-platelets  are  diminished. 

As  far  as  the  blood  is  concerned,  the  chief  points  to 
be  considered  in  diagnosis  are  the  high  color-index  and 
the  presence  of  megaloblasts. 

(2)  Aplastic  Anemia. — The  rare  and  rapidly  fatal 
anemia  which  has  been  described  under  this  name  was 


PLATE  VIII 


J.  W.  Rennell. 
Blood-cells  in  pernicious  anemia.  Xote  variations  in  size  and  shape 
of  the  red  corpuscles;  three  megaloblasts,  one  with  irregular,  deeply 
stained  nucleus;  red  corpuscles  showing  grades  of  polychromato- 
philia,  basophilic  granular  degeneration,  and  one  nuclear  particle;  one 
irritation  leukocyte,  one  lymphocyte,  and  one  polymorphonuclear 
neutrophil.  All  drawn  from  actual  cells  on  two  slides.  Wright's  stain. 
X  800  (i  mm.  =  I  micron). 


SPECIAL  BLOOD    PATHOLOGY  385 

once  considered  a  variety  of  pernicious  anemia,  the 
absence  of  any  attempt  at  blood  regeneration  explain- 
ing the  marked  difference  in  the  blood-picture.  Red 
corpuscles  and  hemoglobin  are  rapidly  diminished  to 
an  extreme  degree.     The  color-index  is  normal  or  low. 

A  B 

Fig.  145. — Red  blood  corpuscles  in  chlorosis  (A)  and  pernicious 
anemia  (C)  contrasted  with  those  of  normal  blood  (B).  In  a  well 
marked  case  of  chlorosis  the  red  corpuscles  are  pale  and  ring-like;  in 
pernicious  anemia  they  are  rich  in  hemoglobin  and  show  marked  vari- 
ations in  size  and  shape.  The  megalocyte  in  the  upper  part  of  the 
figure  is  especially  characteristic  of  pernicious  anemia.  Wright's 
stain.      X  7S0. 

The  leukocyte  count  is  normal  or  low,  with  relative 
increase  of  lymphocytes.  Stained  smears  show  only 
slight  variations  in  size,  shape,  and  staining  properties 
of  the  red  cells.  There  are  no  megaloblasts  and  few 
or  no  normoblasts. 


386  THE   BLOOD 

(3)  Chlorosis. — This  is  probably  a  disease  of  defec- 
tive blood  formation.  It  is  confined  almost  exclusively 
to  unmarried  girls.  The  clinical  symptoms  furnish 
the  most  important  data  for  diagnosis.  The  blood 
resembles  that  of  secondary  anemia  in  many  respects. 

The  most  conspicuous  feature  is  a  marked  decrease  of 
hemoglobin,  accompanied  by  a  slight  decrease  in  number 
of  red  corpuscles.  The  color-index  is  thus  almost  in- 
variably low. 

The  following  figures  represent  about  the  average  for 
well-piarked  cases:  hemoglobin,  40  per  cent.;  red  cor- 
puscles, 4,000,000;  color-index,  0.5.  Much  lower  fig- 
ures are  frequent;  while,  upon  the  other  hand,  mild 
cases  may  show  no  loss  at  all  in  number  of  red  cells. 

As  in  pernicious  anemia,  the  leukocytes  are  normal  or 
decreased  in  number,  with  a  relative  increase  of  lym- 
phocytes. 

In  contrast  to  pernicious  anemia  (and  in  some  degree 
also  to  secondary  anemia),  the  red  cells  are  of  nearly 
uniform  size,  are  pale  (see  Fig.  145),  and  their  average 
diameter  is  somewhat  less  than  normal.  Changes  in 
size,  shape,  and  staining  reactions  occur  only  in  severe 
cases.  Erythroblasts  are  rarely  present.  The  number 
of  platelets  is  generally  decreased. 

(4)  Splenic  Anemia. — This  is  an  obscure  form  of 
anemia  associated  with  great  enlargement  of  the  spleen. 
It  is  probably  a  distinct  entity,  although  several  types 
may  exist.  There  is  decided  decrease  of  hemoglobin 
and  red  corpuscles,  with  moderate  leukopenia  and  rela- 
tive lymphocytosis.  Osier's  15  cases  averaged  47  per 
cent,  hemoglobin  and  3,336,357  red  cells.  Stained  films 
show  notable  irregularities  in  size,  shape,  and  staining 


SPECIAL   BLOOD    PATHOLOGY  387 

properties  only  in  advanced  cases.     Erythroblasts  are 
uncommon. 

B.    Leukemia 

Except  in  rare  instances,  diagnosis  is  easily  made 
from  the  blood  alone,  usually  at  the  first  glance  at  the 
stained  film.  Two  types  of  the  disease  are  commonly 
distinguished:  the  myelogenous  and  the  lymphatic. 
Atypical  cases  are  not  uncommon,  especially  in  children. 
The  disease  is  characterized  by  hyperplasia  of  the 
leukoblastic  bone-marrow  (myelogenous  leukemia)  or  of 
the  lymphoid  tissues  (lymphatic  leukemia),  together 
with  overflow  of  many  immature  leukocytes  and  ex- 
cessive numbers  of  normal  types  into  the  circulating 
blood.  The  more  acute  the  process,  the  more  immature 
are  the  cells  which  appear  in  the  blood. 

1.  Myelogenous  Leukemia  (Plate  IX). — This  is 
usually  a  chronic  disease,  although  acute  cases  have 
been  described. 

Hemoglobin  and  red  corpuscles  show  decided  decrease. 
The  red  count  is  usually  below  3,500,000.  Accurate 
hemoglobin  estimation  is  difficult  because  of  the  great 
number  of  leukocytes.  The  color-index  is  moderately 
low. 

Most  striking  is  the  immense  increase  in  number  of 
leukocytes.  The  count  in  ordinary  cases  varies  between 
100,000  and  400,000.  Counts  over  1,000,000  have  been 
met.  During  spontaneous  remissions,  during  treat- 
ment with  x-ray  or  benzol,  and  during  intercurrent 
infections   the  leukocyte   count   may   fall   to   normal. 

While  these  enormous  leukocyte  counts  are  equaled 
in  no  other  disease,  and  approached  only  in  lymphatic 


388  THE   BLOOD 

leukemia  and  extremely  high-grade  leukocytosis,  the 
diagnosis,  particularly  during  remissions,  depends  more 
upon  qualitative  than  quantitative  changes.  Although 
all  varieties  are  increased,  the  characteristic  and  con- 
spicuous cell  is  the  myelocyte.  This  cell  never  appears 
in  normal  blood;  extremely  rarely  in  leukocytosis;  and 
never  abundantly  in  lymphatic  leukemia.  In  myelog- 
enous leukemia  myelocytes  usually  constitute  more  than 
20  per  cent,  of  all  leukocytes.  Da  Costa's  lowest  case 
gave  7  per  cent.  The  neutrophilic  form  is  generally 
much  more  abundant  than  the  eosinophilic.  Both  show 
considerable  variations  in  size.  Myeloblasts  may  be 
present  in  small  numbers  at  any  stage,  and  in  the  ter- 
minal stages  they  may  be  abundant.  An  increase  in 
their  number  is  of  grave  significance.  Very  constant  in 
myelogenous  leukemia  is  a  marked  absolute,  and  often 
a  relative,  increase  of  eosinophiles  and  basophiles. 
Polymorphonuclear  neutrophiles  and  lymphocytes  are 
absolutely  increased,  although  relatively  decreased. 

The  red  cells  show  the  changes  characteristic  of  a 
severe  secondary  anemia,  except  that  nucleated  reds  are 
commonly  abundant;  in  fact,  no  other  disease  gives  so 
many.  They  are  chiefly  of  the  normoblastic  type. 
Megaloblasts  are  uncommon.  Blood-platelets  are  gen- 
erally increased. 

In  acute  myelogenous  leukemia  the  myeloblast  may 
be  the  predominant  cell,  and  the  blood  will  then  re- 
semble that  of  acute  lymphatic  leukemia.  The  myelo- 
blast can  be  distinguished  from  the  large  lymphocyte 
by  the  oxydase  reaction  (see  p.  342)  although  cases 
occur  in  which  the  type  of  blood  formation  is  so  em- 
bryonic that  the  oxydase  reaction  fails.     As  a  matter 


PLATE  IX 


Fig.  I. — Blood 
of  the  disease: 


ill  lymphatic  leukemia;  X  700.     On  the  left,  chronic  form 
on  the  right,  acute  form  (courtesy  of  Dr.  W.  P.  Harlow). 


\ 


^ 


•'J^YX.A. 


SPECIAL  BLOOD  PATHOLOGY  389 

of  fact  the  test  is  rarely  necessary  for  diagnosis  since 
in  most  cases  of  acute  myelogenous  leukemia  a  sufficient 
number  of  myelocytes  can  be  found  to  put  one  upon  the 
right  track. 

2.  Lymphatic  Leukemia  (Plate  IX). — Chronic 
Form. — There  is  generally  greater  loss  of  hemoglobin 
and  red  corpuscles  than  in  myelogenous  leukemia.  The 
color-index  is  usually  moderately  low. 

The  leukocyte  count  is  high,  but  lower  than  in  the 
myelogenous  type.  Counts  of  100,000  are  about  the 
average,  but  in  many  cases  are  much  lower,  even  as 
low  as  15,000.  Some  cases,  on  the  other  hand,  run  as 
high  as  1,000,000.  This  high  count  is  referable  almost 
wholly  to  increase  of  lymphocytes.  They  generally 
exceed  90  per  cent,  of  the  total  number  and  are  chiefly 
of  the  small  variety.  During  remissions  the  total 
count  may  fall  below  normal,  but  the  percentage  of 
lymphocytes  remains  high.     Myelocytes  are  rare. 

The  red  corpuscles  show  the  changes  usual  in  severe 
secondary  anemia.  Erythroblasts  are  seldom  abun- 
dant.    Blood-platelets  are  decreased. 

Acute  Form. — The  blood  is  similar  to  that  of  the 
chronic  variety.  The  total  leukocyte  count  is  seldom 
so  high,  and  the  large  type  of  lymphocyte  predominates 
in  most  cases.  The  anemia  is  apt  to  be  more  severe 
and  the  normoblasts  more  abundant. 

3.  Anaemia  Infantum  Pseudoleukaemica. — Un- 
der this  name  von  Jaksch  described  a  rare  disease  of 
infancy,  the  proper  classification  of  which  is  uncertain. 
There  is  enlargement  of  liver  and  spleen,  and  some- 
times of  lymph-nodes,  together  with  the  following 
blood    changes:  grave    anemia    with    deformed    and 


39©  THE   BLOOD 

degenerated  red  cells  and  many  erythroblasts  of  both 
normoblastic  and  megaloblastic  types;  great  increase 
in  number  of  leukocytes  (20,000  to  100,000)  and  great 
variations  in  size,  shape,  and  staining  of  leukocytes, 
with  many  atypic  forms,  and  a  few  myelocytes. 

From  the  work  of  more  recent  investigators  it  appears 
probable  that  von  Jaksch's  anemia  is  not  a  distinct  dis- 
ease, and  that  the  reported  cases  have  been  atypical 
forms  of  leukemia,  pernicious  anemia,  or  even  sec- 
ondary anemia  with  leukocytosis.  As  is  well  known,  all 
of  these  conditions  are  apt  to  be  atypical  in  children. 

The  table  on  the  following  page  contrasts  the  dis- 
tinctive blood-changes  in  the  more  common  conditions. 


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SPECIAL  BLOOD   PATHOLOGY  39 1 


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CHAPTER  IV 
THE  STOMACH 

Laboratory  methods  may  be  applied  to  the  diagnosis 
of  stomach  disorders  in:  I.  Examination  of  the  gastric 
contents  removed  with  the  stomach-tube.  II.  Certain 
other  examinations  which  give  information  as  to  the 
condition  of  the  stomach. 

I.  EXAMINATION  OF  THE  GASTRIC  CONTENTS 

Stomach  digestion  consists  mainly  in  the  action  of 
pepsin  upon  proteins  in  the  presence  of  hydrochloric 
acid  and  in  the  curdling  of  milk  by  rennin.  The  fat- 
splitting  ferment,  lipase,  of  the  gastric  juice  has  very 
little  activity  excepting  upon  previously  emulsified  fats 
such  as  those  of  milk  and  egg-yolk. 

Pepsin  and  rennin  are  secreted  by  the  gastric  glands 
as  zymogens — pepsinogen  and  renninogen  respectively 
— which  are  converted  into  pepsin  and  rennin  by  hydro- 
chloric acid.  Hydrochloric  acid  is  secreted  chiefly  by 
the  fundus  end  of  the  stomach.  It  at  once  combines 
loosely  with  the  proteins  of  the  food,  forming  acid- 
metaprotein,  the  first  step  in  protein  digestion.  Hy- 
drochloric acid,  which  is  thus  loosely  combined  with 
proteins,  is  called  "combined"  hydrochloric  acid. 
The  acid  which  is  secreted  after  the  proteins  present 
have  all  been  converted  into  acid-metaprotein  remains 

392 


EXAMINATION   OF   THE    GASTRIC   CONTENTS         393 

as  "free"  hydrochloric  add,  and,  together  with  pepsin, 
continues  the  process  of  digestion. 

At  the  height  of  digestion  the  stomach-contents  con- 
sist essentially  of:  (i)  Water;  (2)  free  hydrochloric  acid; 
(3)  combined  hydrochloric  acid;  (4)  pepsin;  (5)  rennin; 
(6)  mineral  salts,  chiefly  acid  phosphates,  of  no  clinical 
importance;  (7)  particles  of  undigested  and  partly  di- 
gested food;  (8)  various  products  of  digestion  in  solu- 
tion. In  pathologic  conditions  there  may  be  present, 
in  addition,  various  microscopic  structures  and  certain 
organic  acids,  of  which  lactic  acid  is  most  important. 

A  routine  examination  is  conveniently  carried  out  in  the 
following  order: 

1.  Give  the  patient  a  test-meal  upon  an  empty  stomach, 
washing  the  stomach  previously  if  necessary. 

2.  At  the  height  of  digestion,  usually  in  one  hour,  remove 
the  contents  of  the  stomach  with  a  stomach-tube. 

3.  Measure  and  examine  macroscopically. 

4.  Filter.  A  suction  filter  is  desirable,  and  may  be  neces- 
sary when  much  mucus  is  present. 

5.  During  filtration,  examine  microscopically  and  make 
qualitative  tests  for — (a)  free  acids;  (b)  free  hydrochloric 
acid;  (c)  lactic  acid. 

6.  When  sufficient  filtrate  is  obtained,  make  quantitative 
estimations  of — (a)  total  acidity;  (b)  free  hydrochloric  acid; 
(c)  combined  hydrochloric  acid  (if  necessary). 

7.  Make  whatever  additional  tests  seem  desirable,  as  for 
blood,  pepsin,  or  rennin. 

A.  Obtaining  The  Contents 

Gastric  juice  is  secreted  continuously,  but  quantities 
sufl&ciently  large   for  examination  are   often  not  ob- 


394  THE    STOMACH 

tainable  from  the  fasting  stQmach.  ,  In  clinical  work, 
therefore,  it  is  desirable  to  stimulate  secretion  with 
food — which  is  the  natural  and  most  efficient  stimulus — 
before  attempting  to  collect  the  gastric  fluid.  Dif- 
ferent foods  stimulate  secretion  to  diflferent  degrees, 
hence  for  the  sake  of  uniform  results  certain  standard 
*' test-meals"  have  been  adopted. 

I.  Test=mea!s. — It  is  customary  to  give  the  test- 
meal  in  the  morning,  since  the  stomach  is  most  apt  to  be 
empty  at  that  time.  If  it  be  suspected  that  the  stomach 
will  not  be  empty,  it  should  be  washed  out  with  water 
the  evening  before. 

(i)  Ewald's  test-breakfast  consists  of  a  roll  (or  two 
slices  of  bread),  without  butter,  and  two  small  cups 
(300  to  400  c.c.)  of  water,  or  weak  tea,  without  cream  or 
sugar.  It  should  be  well  masticated.  The  contents  of 
the  stomach  are  to  be  removed  one  hour  afterward, 
counting  from  the  beginning,  not  the  end,  of  the  meal. 
This  test-meal  has  long  been  used  for  routine  exami- 
nations. Its  disadvantage  is  that  it  introduces,  with 
the  bread,  a  variable  amount  of  lactic  acid  and  numerous 
yeast-cells.  This  source  of  error  may  be  eliminated 
by  substituting  a  shredded  whole-wheat  biscuit  for  the 
roll.  The  shredded  wheat  test-meal  is  now  widely  used 
and  is  probably  the  most  satisfactory  for  general 
purposes. 

(2)  Boas'  test-breakfast  consists  of  a  tablespoonful 
of  rolled  oats  in  a  quart  of  water,  boiled  to  one  pint,  with 
a  pinch  of  silt  added.  It  should  be  withdrawn  in  forty- 
five  minutes  to  one  hour.  This  meal  does  not  contain 
lactic  acid,  and  is  usually  given  when  the  detection  of 
lactic  acid  is  important,  as  in  suspected  gastric  cancer. 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        395 

The  stomach  should  always  be  washed  with  water  the 
evening  previous. 

(3)  Riegel's  test-meal  consists  of  400  c.c.  of  bouillon, 
a  broiled  beefsteak  (about  150-200  Gm.),  and  150  Gm. 
of  mashed  potato.  Since  it  tends  to  clog  the  tube,  it 
must  be  thoroughly  masticated. 

(4)  Fischer's  test-meal  is  similar,  but  probably  pref- 
erable. It  consists  of  an  Ewald  breakfast  plus  about 
}''i  pound  lean,  finely  chopped  Hamburger  steak, 
broiled,  and  lightly  seasoned.  This  and  Riegel's  may 
be  removed  in  three  to  four  hours.  They  give  some- 
what higher  acidity  values  than  the  Ewald  breakfast. 

2.  Withdrawal  of  the  Contents. — The  Boas  stom- 
ach-tube, with  bulb,  is  the  form  which  is  most  widely 
used.  It  should  be  of  rather  large  caliber,  and  have  an 
opening  in  the  tip  and  one  or  two  in  the  side  near  the 
tip.  When  not  in  use  it  may  be  kept  in  a  vessel  of 
borax  solution,  and  should  be  well  washed  in  hot  water 
both  before  and  after  using. 

It  is  important  confidently  to  assure  the  patient  that 
introduction  of  the  tube  cannot  possibly  harm  him;  and 
that  if  he  can  control  the  spasm  of  his  throat,  he  will 
experience  very  little  choking  sensation.  When  pa- 
tients are  very  nervous  it  is  well  to  spray  the  throat 
with  cocain  solution. 

The  tube  should  be  dipped  in  warm  water  just  before 
using;  the  use  of  glycerin  or  other  lubricant  is  undesir- 
able. With  the  patient  seated  upon  a  chair,  his  cloth- 
ing protected  by  towels  or  a  large  apron,  and  his  head 
tilted  forward,  the  tip  of  the  tube,  held  as  one  would  a 
pen,  is  introduced  far  back  into  the  pharynx.  He  is 
then  urged  to  swallow,  and  the  tube  is  pushed  boldly 


396  THE    STOMACH 

into  the  esophagus  until  the  ring  upon  it  reaches  the 
incisor  teeth,  thus  indicating  that  the  tip  is  in  the  stom- 
ach. If,  now,  the  patient  cough  or  strain  as  if  at  stool, 
the  contents  of  the  stomach  will  usually  be  forced  out 
through  the  tube.  Should  it  fail,  the  fluid  can  generally 
be  pumped  out  by  alternate  compression  of  the  tube 
and  the  bulb.  If  unsuccessful  at  first,  the  attempts 
should  be  repeated  with  the  tube  pushed  a  little  further 
in,  or  withdrawn  a  few  inches,  since  the  distance  to  the 
stomach  is  not  the  same  in  all  cases.  The  tube  may 
become  clogged  with  pieces  of  food,  in  which  case  it 
must  be  withdrawn,  cleaned,  and  reintroduced.  If, 
after  all  efforts,  no  fluid  is  obtained,  another  test-meal 
should  be  given  and  withdrawn  after  a  somewhat  shorter 
period,  since,  owing  to  excessive  motility,  the  stomach 
may  empty  itself  in  less  than  the  usual  time. 

Care  must  be  exercised  to  prevent  saliva  running  down 
the  outside  of  the  tube  and  mingling  with  the  gastric 
juice  in  the  basin. 

As  the  tube  is  removed,  it  should  be  pinched  between 
the  fingers  so  as  to  save  any  fluid  that  may  be  in  it. 

The  stomach-tube  must  be  used  with  great  care,  or 
not  at  all,  in  cases  of  gastric  ulcer,  aneurysm,  uncom- 
pensated heart  disease,  and  marked  arteriosclerosis. 
Except  in  gastric  ulcer,  the  danger  hes  in  the  retching 
produced,  and  the  tube  can  safely  be  used  if  the  patient 
takes  it  easily. 

The  above  description  applies  to  the  use  of  the  type 
of  stomach  tube  which  for  many  years  has  been  the 
standard.  The  procedure  is  made  much  easier  both 
for  physician  and  patient  by  use  of  the  new  Rehfuss 
stomach  tube.     This  is  a  modification  of  Einhorn's 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        397 

duodenal  tube  and  consists  of  a  slender  rubber  tube 
with  perforated  olive-shaped  metal  tip.  When  the  tube 
is  to  be  introduced,  the  metal  tip  is  pushed  well  back  into 
the  patient's  pharynx  and  it  is  then  easily  swallowed. 
After  it  has  reached  the  stomach  the  heavy  tip  sinks 
to  the  most  dependent  portion  and  as  much  or  as 
little  of  the  stomach  contents  as  is  desired  may  be 
drawn  off  by  means  of  an  aspirating  syringe.  The 
tube  is  especially  useful  for  the  fractional  method  of 

Time  in  hours  and  minutes 
.      ,  -   F.5.    0-15  0-30  0-45  I-OO   I-I5    1-30    US  2-00  2-I5   Z-30  2-45 

40 

1 20 
10 
0 

Fig.  146. — Diagram  showing  the  average  acidity  of  stomach  fluid 
of  24  healthy  persons  studied  by  Talbot  by  the  fractional  method; 
F.S.,  fasting  stomach. 


- 

cr' 
f 

,--0-, 

1        •        1        I 

■~°N^^-Total  Acidity 

(/_ 

' 

^•0— 

P'' 

-^ 

y>~~~ 

-^ 

A 

^0— 

-^>, 

■>D 

a.^ 

-cf' 

y 

^V              A 

f  ^  Vraa  h 

cidit 

y 

\ 

V-      1 

ICC  rt 

- 

^ 

examination  described  below,  as  it  may  be  left  in  place 
for  a  long  time  without  serious  inconvenience  to  the 
patient. 

With  the  practical  appreciation  that  there  is  great  varia- 
tion in  the  time  at  which  the  height  of  digestion  is  reached, 
a  new  method  of  examination  known  as  the  "fractional 
method"  has  come  into  wide  use.  This  is  carried  out  as 
follows: 

I.  Insert  a  Rehfuss  stomach-tube  before  breakfast  and 
empty  the  stomach  as  far  as  possible. 


398  THE    STOMACH 

2.  Remove  the  tube  and  give  an  Ewald  test-breakfast, 
which  must  be  chewed  thoroughly. 

3.  Re-insert  the  tube  and  withdraw  5  c.c.  01  the  stomach 
content  at  fifteen-minute  intervals  until  the  fluid  is  free 
from  food  particles  or  until  the  acidity  has  returned  to  the 
same  level  as  was  found  in  the  fasting  content.  The  tube 
is  left  in  place  during  the  whole  procedure.  Ordinarily  it 
causes  very  little  nausea. 

4.  Examine  each  of  the  5-c.c.  portions  and  also  the  fluid 
from  the  fasting  stomach  for  total  acidity,  free  hydrochloric 
acid  and  lactic  acid. 

By  means  of  the  Rehfuss  tube  a  much  larger  quantity  of 
gastric  juice  can  often  be  obtained  from  the  fasting  stomach' 
than  was  formerly  believed  possible.  The  quantity  is  very 
variable,  ranging  from  5  c.c.  to  150  c.c.  or  even  more,  and 
averaging  about  45  c.c.  The  acidity  values  are  also  vari- 
able. Averages  for  the  fasting  content  and  each  of  the 
fifteen-minute  periods  are  shown  in  Fig.  146. 

B.  Physical  Examination 

Under  normal  conditions  50  to  100  c.c.  of  fluid  can  be 
obtained  one  hour  after  administering  Ewald's  break- 
fast. Larger  amounts  point  to  motor  insufficiency 
or  hypersecretion;  less  than  20  c.c,  to  too  rapid  empty- 
ing of  the  stomach,  or  else  to  incomplete  removal. 
Upon  standing,  it  separates  into  two  layers:  the  lower 
consisting  of  particles  of  food;  the  upper,  of  an  almost 
clear,  faintly  yellow  fluid.  The  extent  to  which  diges- 
tion has  taken  place  can  be  roughly  judged  from  the 
appearance  of  the  food-particles. 

The  reaction  is  frankly  acid  in  health  and  in  nearly 
all  pathologic  conditions.  It  may  be  neutral  or  slightly 
alkaline  in  some  cases  of  gastric  cancer  and  marked 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        309 

chronic  gastritis,  or  when  contaminated  by  a  consider- 
able amount  of  saliva. 

A  small  amount  of  mucus  is  present  normally. 
Large  amounts,  when  the  gastric  contents  are  obtained 
with  the  tube  and  not.  vomited,  point  to  chronic  gas- 
tritis. Mucus  is  recognized  from  its  characteristic 
sfimy  appearance  when  the  fluid  is  poured  from  one 
vessel  into  another.  It  is  more  frequently  seen  in 
stomach  washings  than  in  the  fluid  removed  after  a 
test-meal. 

A  trace  of  bile  is  common  as  a  result  of  excessive 
straining  while  the  tube  is  in  the  stomach.  Large 
amounts  are  very  rarely  found,  and  generally  point  to 
obstruction  in  the  duodenum.  Bile  produces  a  yellow- 
ish or  more  frequently  greenish  discoloration  of  the 
fluid. 

Blood  is  often  recognized  by  simple  inspection,  but 
more  frequently  requires  a  chemic  test.  It  is  bright  red  ' 
when  very  fresh,  and  dark,  resembling  coffee-grounds, 
when  older.  Vomiting  of  blood,  or  hemalemesis ,  may  be 
mistaken  for  pulmonary  hemorrhage,  or  hemoptysis.  In 
the  former  the  fluid  is  acid  in  reaction  and  usually  dark 
red  or  brown  in  color  and  clotted,  while  in  hemoptysis 
it  is  brighter  red,  frothy,  alkaline,  and  usually  mixed 
with  a  variable  amount  of  mucus.  When  the  blood 
is  small  in  amount  and  bright  red  the  possibility  that 
it  originates  from  injury  done  by  the  tube  must  not  be 
overlooked. 

Particles  of  food  eaten  hours  or  even  days  previously 
may  be  found,  and  indicate  deficient  motor  power. 

Search  should  always  be  made  for  bits  of  tissue  from 
the  gastric  mucous  membrane  or  new  growths.     These, 


400  THE   STOMACH 

whai  examined  by  a  p)atfaoIogist,  will  sometimes  render 
the  diagnosis  dear. 

C  Chexhc  Exahdiatioii 

A  routine  chemic  examination  of  the  gastric  contents 
involves  qualitative  tests  for  free  acids,  f re«  hydrochloric 
acid,  and  organic  acids,  and  quantitative  estimations  of 
total  acidity,  firee  hj^tirochloric  acid,  and  sometimes 
combined  hydrochloric  acid.  Other  tests  are  applied 
when  indicated. 

1.  Qualitative  Tests, — (i)  Free  Acids. — Thepres- 
a»ce  or  absence  of  free  acids,  without  reference  to  the 
kind,  is  easily  determined  by  means  of  Congo-red, 
although  the  test  is  not  much  used  in  {Huctice. 

Congo-red  Test. — Take  a  few  drq[»  of  a  strong  akoholic 
solution  of  Congo-red  in  a  test-tube,  dilute  with  wato*  to  a 
stioi^  red  c(d<x^,  and  add  a  few  cubic  centimeters  of  filtered 
^Btric  juice.  The  af^tearanoe  of  a  bltie  coler  shows  ihe 
piesence  of  sune  free  acid  (Plate  X,  B,  BO.  Since  the  test 
is  moce  sensitive  to  minoal  than  to  organic  adds,  a  marked 
reaction  points  to  the  presence  of  free  hydrochkKic  add. 

Thick  filter-paper  soaked  in  Coogo-red  solution,  dried, 
and  cut  into  strips  may  be  used,  but  the  test  is  much  less 
delicate  whoi  thus  a]^£ed. 

(2)  Free  Hydrochloric  Add. — In  addition  to  its  digest- 
K-e  functicm,  free  hydrochloric  acid  is  an  efficient  anti- 
septic It  preN-ents  or  retards  fermentation  and  lactic- 
acid  formation,  and  is  an  important  means  of  protection 
against  the  entrance  of  path<^«w:  (Hganisms  into  the 
body-.     It  is  nevo*  absoit  in  health. 

DimedijljuDiidoazobenzol  Test — ^To  a  little  of  the  fil- 
tered gastric  juice  m  a  test-tube,  or  to  several  dn^  in  a 


PLATE  X 


..MalCBDrtiHI. 


EXAMINATION    OF    THE    GASTRIC   CONTENTS         4OI 

porcelain  dish,  add  a  drop  of  0.5  per  cent,  alcoholic  solution 
of  dimethylamidoazobenzol.  In  the  presence  of  free  hy- 
drochloric acid  there  will  at  once  appear  a  cherry-red  color, 
varying  in  intensity  with  the  amount  of  acid  (Plate  XI,  C). 
This  test  is  very  delicate;  but,  unfortunately,  organic  acids, 
when  present  in  large  amounts  (above  0.5  per  cent.),  give  a 
similar  reaction.  The  color  obtained  with  organic  acids  is, 
however,  more  of  an  orange  red. 

Boas'  Test. — This  test  is  less  delicate  than  the  preceding, 
but  is  more  reliable,  since  it  reacts  only  to  free  hydrochloric 
acid.     It  is  probably  the  best  routine  test. 

In  a  porcelain  dish  mix  a  few  drops  of  the  gastric  juice  and 
the  reagent,  and  slowly  evaporate  to  dryness  over  a  flame, 
taking  care  not  to  scorch.  The  appearance  of  a  rose-red  color, 
which  fades  upon  cooling,  shows  the  presence  of  free  hydro- 
chloric acid  (Plate  X,  i). 

Boas^  reagent  consists  of  5  Gm.  resublimed  resorcinol,  and 
3  Gm.  cane-sugar,  in  100  c.c.  alcohol.  The  solution  keeps 
well,  which,  from  the  practitioner's  view-point,  makes  it 
preferable  to  Giinzburg's  phloroglucin-vanillin  reagent 
(phloroglucin,  2  Gm.;  vanilhn,  i  Gm.;  absolute  alcohol, 
30  c.c).  The  latter  is  just  as  delicate,  is  applied  in  the 
same  way,  and  gives  a  sharper  reaction  (Plate  X,  2),  but 
is  unstable. 

(3)  Organic  Acids.— Lactic  acid  is  the  most  common, 
and  is  taken  as  the  type  of  the  organic  acids  which 
appear  in  the  stomach-contents.  It  is  a  product  of 
bacterial  activity.  Acetic  and  butyric  acids  are  some- 
times present.  Their  formation  is  closely  connected 
with  that  of  lactic  acid,  and  they  are  rarely  tested  for. 
When  abundant,'  they  may  be  recognized  by  their  odor 
upon  heating.  Butyric  acid  gives  the  odor  of  rancid 
butter. 

26 


402  THK    STOMACH 

Lactic  acid  is  never  present  at  the  height  of  digestion 
in  health.  Although  usually  present  early  in  digestion, 
it  disappears  when  free  hydrochloric  acid  begins  to 
appear.  Small  amounts  may  be  introduced  with  the 
food.  Pathologically,  small  amounts  may  be  present 
whenever  there  is  stagnation  of  the  gastric  contents  with 
deficient  hydrochloric  acid,  as  in  many  cases  of  dilata- 
tion of  the  stomach  and  chronic  gastritis.  The  presence 
of  notable  amounts  of  lactic  acid  (more  than  o.i  per 
cent,  by  Strauss'  test)  is  strongly  suggestive  of  gastric 
cancer,  and  is  probably  the  most  valuable  single  symp- 
tom of  the  disease. 

As  already  stated,  the  Ewald  test-breakfast  intro- 
duces a  small  amount  of  lactic  acid,  but  rarely  enough 
to  respond  to  the  tests  given  here.  In  every  case, 
however,  in  which  its  detection  is  important,  the 
shredded-wheat  biscuit  or  Boas'  test-breakfast  should 
be  given,  the  stomach  having  been  thoroughly  washed 
the  evening  before. 

UfEelmann's  Test  for  Lactic  Acid. — Thoroughly  shake 
up  5  c.c.  of  filtered  stomach  fluid  with  50  c.c.  of  ether  for  at 
least  ten  minutes.  Collect  the  ether  and  evaporate.  Dis- 
solve the  residue  in  5  c.c.  of  water  and  test  with  Uffelmann's 
reagent  as  follows: 

In  a  test-tube  mix  3  drops  concentrated  solution  of  phenol 
and  3  drops  saturated  aqueous  solution  of  ferric  chlorid. 
Add  water  until  the  mixture  assumes  an  amethyst-blue 
color.  To  this  add  the  solution  to  be  tested.  The  appear- 
ance of  a  canary-yellow  color  indicates  the  presence  of  lactic 
acid  (Plate  X,  A,  A'). 

Uffelmann's  test  may  be  applied  directly  to  the  stomach- 
contents  without  extracting  with  ether,  but  is  then  neither 


EXAMINATION    OF   THE    GASTRIC    CONTENTS         403 


25l 


sensitive  nor  reliable,  because  of  the  phosphates,  sugars,  and 
other  interfering  substances  which  may  be  present. 

Kelling's  Test  (Simon's  Modification). — This  is  much 
more  satisfactory  than  UfTelmann's.  To  a  test-tube  of 
distilled  water  add  sufficient  ferric  chlorid  solution  to  give  a 
faint  yellowish  tinge.  Pour  half  of  this  into  a  second  test- 
tube  to  serve  as  a  control.  To  the  other  add  a  small  amount 
of  the  gastric  juice.  Lactic  acid  gives  a 
distinct  yellow  color  which  is  readily  rec- 
ognized by  comparison  with  the  control. 

Strauss'  Test  for  Lactic  Acid.^ — This  is 
a  good  test  for  clinical  work,  since  it  gives 
a  rough  idea  of  the  quantity  present  and 
is  not  sufficiently  sensitive  to  respond  to 
the  traces  of  lactic  acid  which  some  test- 
meals  introduce.  Strauss'  instrument 
(Fig.  147)  is  essentially  a  separatory 
funnel  with  a  mark  at  5  c.c.  and  one  at 
25  c.c.  Fill  to  the  5-c.c.  mark  with 
filtered  stomach  fluid,  and  to  the  2 5-c.c. 
mark  with  ether.  Shake  thoroughly  for 
ten  or  fifteen  minutes,  let  stand  until  the 
ether  separates,  and  then,  by  opening  the 
stop-cock,  allow  the  gastric  juice  to  run 
out.  Fill  to  the  25-c.c.  mark  with  water,  pj^  147— Sepa- 
and  add  2  drops  of  a  10  per  cent,  solution  ratory    funnel    for 

r    r       •        VI      -J        ou    1  i.1  rc  Strauss'     lactic-acid 

of  ferric  chlorid.     Shake  gently.     If  o.i  ^^^^  (Sahii) 

per  cent,  or  more  lactic  acid  be  present, 

the  water  will  assume  a  strong  greenish-yellow  color.     A 

slight  tinge  will  appear  with  0.05  per  cent. 

(4)  Pepsin  and  Pepsinogen. — Pepsinogen  itself  has 
no  digestive  power.  It  is  secreted  by  the  gastric  glands, 
and  is  transformed  into  pepsin  by  the  action  of  a  free 
acid.     Although  pepsin   digests  proteins  best  in   the 


404  THE    STOMACH 

presence  of  free  hydrochloric  acid,  it  has  a  slight  digest- 
ive acti\-ity  in  the  presence  of  organic  or  combined 
hydrochloric  acids. 

The  amount  is  not  influenced  by  neuroses  or  circula- 
tory disturbances.  Absence  or  marked  diminution, 
therefore,  indicates  organic  disease  of  the  stomach. 
This  is  an  important  p>oint  in  diagnosis  between 
functional  and  organic  conditions.  Pepsin  is  rarely  or 
never  absent  in  the  presence  of  free  hydrochloric 
acid. 

Test  for  Pepsin  and  Pepsinogen. — With  a  cork-borer  cut 
small  cylinders  from  the  coagulated  white  of  an  egg,  and  cut 
these  into  disks  of  uniform  size.  The  egg  should  be  cooked 
very  slowly,  preferably  over  a  water-bath,  so  that  the  white 
may  be  readily  digestible.  The  disks  may  be  preserved  in 
glycerin,  but  must  b«  washed  in  water  before  using. 

Place  a  disk  in  each  of  three  test-tubes. 

Into  tube  No.  i  put  lo  c.c.  distilled  water,  5  gr.  pepsin, 
U.  S.  P.,  and  3  drops  of  the  oflficial  dilute  hydrochloric  acid. 

Into  tube  No.  2  put  10  c.c.  filtered  gastric  juice. 

Into  tube  No.  3  put  10  c.c.  filtered  gastric  juice  and  3  drops 
dilute  hydrochloric  acid. 

Place  the  tubes  in  an  incubator  or  in  warm  water  for 
three  hours  or  longer.  At  intervals  obser\'e  the  extent  to 
which  the  egg-albumen  has  been  digested.  This  is  recog- 
nized by  the  depth  to  which  the  dbk  has  become  translucent. 

Tube  No.  I  is  used  for  comparison,  and  should  show  the 
eflFect  of  normal  gastric  juice. 

Digestion  of  the  egg  in  tube  No.  2  indicates  the  presence 
of  both  pepsin  and  free  hydrochloric  acid. 

When  digesdon  fails  in  tube  No.  2  and  occurs  in  No.  3, 
pepsinogen  is  present,  having  been  transformed  into  pepsin 
by  the  hydrochloric  acid  added.  Should  digestion  fail  in 
this  tube,  both  pepsin  and  jjepsinogen  are  absent. 


EXAMINATION    OF   THE    GASTRIC    CONTENTS        405 

(5)  Rennin  and  Renninogen. — Rennin  is  the  milk- 
curdling  ferment  of  the  gastric  juice.  It  is  derived  from 
renninogen  through  the  action  of  hydrochloric  acid. 
Deficiency  of  rennin  has  the  same  significance  as 
deficiency  of  pepsin,   and   is  more  easily  recognized. 

Test  for  Rennin. — Neutralize  5  c.c.  filtered  gastric  juice 
with  very  dilute  sodium  hydroxid  solution;  add  5  c.c.  fresh 
milk,  and  place  in  an  incubator  or  in  a  vessel  of  water  at 
about  4o°C.  Coagulation  of  the  milk  in  ten  to  fifteen 
minutes  shows  a  normal  amount  of  rennin.  Delayed  coagu- 
lation denotes  a  less  amount. 

(6)  Peptid-splitting  Enzyme. — It  has  been  found  that 
in  cancer  of  the  stomach  there  may  be  present  a  patho- 
logic ferment  which  is  capable  of  splitting  peptids  into 
amino-acids.  No  such  ferment  is  present  normally, 
the  gastric  juice  being  incapable  of  carrying  digestion 
to  the  amino-acid  stage.  Neubauer  and  Fischer  have 
utilized  this  fact  for  the  diagnosis  of  gastric  cancer 
by  subjecting  the  dipeptid,  glycyl-tryptophan,  to  the 
action  of  the  gastric  fluid  and  testing  for  the  presence 
of  the  amino-acid  tryptophan.  The  method  is  as 
follows : 

Place  10  c.c.  of  the  filtered  gastric  juice  and  about  i  c.c. 
of  glycyl-tryptophan  in  a  test-tube,  overlay,  with  toluol  to 
prevent  bacterial  action,  and  place  in  an  incubator  at  about 
38°C.  At  the  end  of  twenty-four  hours  pipet  off  a  few  cubic 
centimeters  and  test  for  tryptophan  as  follows:  Acidify  with 
a  few  drops  of  3  per  cent,  acetic  acid,  add  a  very  httle  bromin 
vapor  with  a  medicine-dropper,  and  shake.  The  appear- 
ance of  a  rose-red  color  shows  the  presence  of  tryptophan 
and  hence  of  the  peptid-splitting  ferment.  The  color 
quickly  disappears  if  too  much  bromin  is  added.     If  no 


4o6  THE    STOMACH 

color  appears  at  first,  add  more  bromin  vapor  in  small 
quantities.  -  Only  when  the  fluid  has  become  yellow  from 
excess  of  bromin  can  the  test  be  considered  negative. 

Before  applying  this  method,  the  stomach  fluid  must  be 
tested  for  pre-exisjting  tryptophan,  blood  (see  p.  407),  and 
bile  (see  p.  179).  Blood  and  pancreatic  juice  each  contain 
peptid-splitting  ferments,  and  pancreatic  juice  may  be 
assumed  to  be  present  if  bile  is  detected. 

Glycyl-tryptophan  can  be  purchased  in  bottles,  each 
containing  a  httle  toluol  and  the  correct  amount  of  the  di- 
f)eptid  for  one  test.  The  gastric  juice  is  introduced  into  the 
bottle  to  the  level  of  a  mark  on  its  side  and  then  incubated. 
Such  an  outfit  is  called  a  "ferment  diagnosticum." 

Instead  of  glycyl-tryptophan,  Jacque  and  Woodyatt  and 
others  have  used  20  c.c.  sterilized  filtered  2  per  cent,  solu- 
tion of  Witte's  peptone  for  each  10  c.c.  of  stomach  fluid. 
They  then  estimate  amino-acids  in  10  c.c.  of  the  mixture 
before  incubating  and  in  10  c.c.  afterward,  using  the  formalin 
method  which  is  given  for  ammonia  in  urine  (see  p.  147). 
The  difference  between  the  two  estimations  expresses  the 
degree  of  peptolysis. 

The  value  of  the  test  is  impaired  by  Warfield's  dis- 
covery of  peptid-splitting  ferments  in  the  saliva.  Later 
workers  have  shown  that  much,  at  least,  of  the  pepto- 
lytic  activity  of  the  saliva  is  due  to  ferments  of  leuko- 
cytes and  bacteria,  which  are  capable  of  splitting 
proteins  as  well  as  peptids.  The  chief  source  of  error, 
however,  appears  to  be  regurgitated  trypsin,  which 
may  be  present  in  the  absence  of  bile.  To  exclude 
these  sources  of  error  Friedman  and  Hamburger  pro- 
pose a  control  test  for  proteolytic  ferments,  using 
edestin  as  substrate.  If  the  edestin  test  is  positive, 
the  glycyl-tryptophan  test  cannot  be  relied  upon. 
It  is  performed  as  follows: 


EXAMINATION   OF   THE    GASTRIC   CONTENTS         407 

Edestin  Test. — The  gastric  juice  is  filtered,  neutralized 

with  normal  Na2C03  solution,  using  phenolphthalein  as 

N 
indicator,  and  then  brought  to  an  alkalinity  equal  to  — ^— 

Na2C03,  in  order  to  inactivate  pepsin.  Place  2  c.c.  of  a 
0.1  per  cent,  solution  of  edestin^  in  o.i  per  cent.  NaaCOs  in 
each  of  four  test-tubes.  To  three  tubes  add  2  c.c,  i  c.c, 
and  0.5  c.c.  of  the  faintly  alkalinized  gastric  fluid,  reserving 
the  fourth  tube  as  a  control  and  adding  to  it  only  a  drop  of 
phenolphthalein  solution.  Place  the  four  tubes  in  an  incu- 
bator at  37°C.  At  the  end  of  four  hours  exactly  neutralize 
the  contents  of  each  of  the  tubes  with  5  per  cent,  acetic  acid. 
When  the  neutral  point  is  reached  all  the  undigested  edestin 
will  be  precipitated.  The  degree  of  digestion  is  indicated  by 
the  amount  of  turbidity  compared  with  that  in  the  control 
tube.     Absence  of  turbidity  denotes  complete  digestion. 

(7)  Blood  is  present  in  the  vomitus  in  a  great  variety 
of  conditions.  When  found  in  the  fluid  removed  after 
a  test-meal,  it  commonly  points  toward  ulcer  or  car- 
cinoma. Blood  can  be  detected  in  nearly  one-half  of 
the  cases  of  gastric  cancer.  The  presence  of  swallowed 
blood  and  blood  from  injury  done  by  the  stomach-tube 
must  be  excluded. 

Test  for  Blood  in  Stomach-contents. — Extract  with  ether 
to  remove  fat.  To  10  c.c.  of  the  fat-free  fluid  add  3  or 
4  c.c  of  glacial  acetic  acid  and  shake  the  mixture  thor- 
oughly with  about  5  c.c.  of  ether.  Separate  the  ether  and 
use  half  of  it  for  the  guaiac  or  benzidin  test  (see  p.  181). 
In  the  case  of  a  positive  reaction  the  remainder  of  the 
ether-extract  may  be  examined  spectroscopically  after 
treating  so  as  to  develop  the  bands  of  hemochromogen 
(see  pp.  369,  371). 

^  Edestin  is  a  protein  extracted  from  hemp  seed.  It  can  be 
purchased  from  Eimer  and  Amend,  New  York. 


408  THE    STOMACH 

When  brown  particles  are  present  in  the  fluid,  the  hemin 
test  should  be  applied  directly  to  them. 

2.  Quantitative  Tests.— (i)  Total  Acidity.— The 
acid-reacting  substances  which  contribute  to  the  total 
acidity  are  free  hydrochloric  acid,  combined  hydro- 
chloric acid,  acid  salts,  mostly  phosphates,  and,  in  some 
pathologic  conditions,  the  organic  acids.  The  total 
acidity  is  normally  about  50  to  75  degrees  (see  method 
below),  or,  when  estimated  as  hydrochloric  acid,  about 
0.2  to  0.3  per  cent.  With  Riegel's  or  Fischer's  test- 
meal  the  figures  are  a  little  higher. 

Topfer's  Method  for  Total  Acidity. — In  an  evaporating 
dish  or  small  beaker  (an  "after-dinner"  coffee-cup  is  a  very 
convenient  substitute)  take  10  c.c.  filtered  stomach-contents 
and  add  3  or  4  drops  of  the  indicator,  a  i  per  cent,  alcoholic 
solution  of  phenolphthalein.  When  the  quantity  of 
stomach  fluid  is  small,  5  c.c.  may  be  used,  but  results  are  less 
accurate  than  with  a  larger  amount.  Add  decinormal  solu- 
tion of  sodium  hydroxid  drop  by  drop  from  a  buret,  until 
the  fluid  assumes  a  rose-red  color  which  does  not  become 
deeper  upon  addition  of  another  drop  (Plate  XI,  A,  A'). 
In  ordinary  titrations  the  end-point  is  the  appearance  of  the 
first  permanent  pink,  but  owing  to  interaction  of  phosphates 
it  is  advised  (Wood)  to  carry  the  titration  of  gastric  juice 
a  little  farther,  as  here  indicated.  When  this  point  is 
reached,  all  the  acid  has  been  neutralized.  The  end  reac- 
tion will  be  sharper  if  the  fluid  be  saturated  with  sodium 
chlorid.  A  sheet  of  white  paper  beneath  the  beaker  facili- 
tates recognition  of  the  color  change. 

In  clinical  work  the  amount  of  acidity  is  expressed  by  the 
number  of  cubic  centimeters  of  the  decinormal  sodium  hy- 
droxid solution  which  would  be  required  to  neutralize  100 
c.c.  of  the  gastric  juice,  each  cubic  centimeter  representing 


EXAMINATION    OF    THE    GASTRIC    CONTENTS         409 

one  degree  of  acidity.  Hence,  multiply  the  number  of  cubic 
centimeters  of  decinormal  solution  required  to  neutralize 
the  10  c.c.  of  stomach  fluid  by  10.  This  gives  the  number  of 
degrees  of  acidity.  The  amount  may  be  expressed  in  terms 
of  hydrochloric  acid,  if  one  remembers  that  each  degree  is 
equivalent  to  0.00365  per  cent,  hydrochloric  acid.  Some 
one  suggests  that  this  is  the  number  of  days  in  the  year,  the 
last  figure,  5,  indicating  the  number  of  decimal  places. 

Example. — Suppose  that  7  c.c.  of  decinormal  solution 
were  required  to  bring  about  the  end  reaction  in  10  c.c.  gas- 
tric juice;  then  7  X  10  =  70  degrees  of  acidity;  and,  ex- 
pressed in  terms  of  hydrochloric  acid,  70  X  0.00365  =  0.255 
per  cent. 

Preparation  of  decinormal  solutions  is  described  in  text- 
books on  chemistry.  The  practitioner  will  find  it  best  to 
have  them  made  by  a  chemist,  or  to  purchase  from  a  chemic 
supply  house.  Preparation  of  an  approximately  decinormal 
solution  is  described  on  page  652. 

(2)  Hydrochloric  Acid.— After  the  Ewald  and  Boas 
test-breakfasts  the  amount  of  free  hydrochloric  acid 
varies  normally  between  25  and  50  degrees,  or  about  o.i 
to  0,2  per  cent.  In  disease  it  may  go  considerably 
higher  or  may  be  absent  altogether. 

When  the  amount  of  free  hydrochloric  acid  is  normal, 
organic  disease  of  the  stomach  probably  does  not  exist. 

Increase  of  free  hydrochloric  acid  above  50  degrees 
Qtyperchlorhydria)  generally  indicates  a  neurosis,  but 
also  occurs  in  most  cases  of  gastric  ulcer  and  beginning 
chronic  gastritis. 

Decrease  of  free  hydrochloric  acid  below  25  degrees 
(hypochlorhydria)  occurs  in  some  neuroses,  chronic  gas- 
tritis, early  carcinoma,  pellagra,  and  most  conditions 
associated  with  general  systemic  depression.     Marked 


4IO  THE    STOMACH 

variation  in  the  amount  at  successive  examinations 
strongly  suggests  a  neurosis.  Too  low  values  are  often 
obtained  at  the  first  examination,  the  patient's  dread 
of  the  introduction  of  the  tube  probably  inhibiting 
secretion. 

Absence  of  free  hydrochloric  acid  {ac/dorhydria)  occurs 
in  most  cases  of  gastric  cancer  and  far-advanced  chronic 
gastritis,  in  many  cases  of  pernicious  anemia  and 
pellagra,  and  sometimes  in  hysteria  and  pulmonary 
tuberculosis. 

The  presence  of  free  hydrochloric  acid  presupposes  a 
normal  amount  of  combined  hydrochloric  acid,  hence 
the  combined  need  not  be  estimated  when  the  free  acid 
has  been  found.  When,  however,  free  hydrochloric  acid 
is  absent,  it  is  important  to  know  whether  any  acid  is 
secreted,  and  an  estimation  of  the  combined  acid  then 
becomes  of  great  value.  The  normal  average  after  an 
Ewald  breakfast  is  about  lo  to  15  degrees,  the  quantity 
depending  upon  the  amount  of  protein  in  the  test-meal. 
Somewhat  higher  figures  are  obtained  after  a  Riegel  or 
Fischer  test-meal. 

Topfer's   Method   for  Free   Hydrochloric   Acid. — In   a 

beaker  take  10  c.c.  filtered  stomach  fluid  and  add  4  drops 
of  the  indicator,  a  0.5  per  cent,  alcoholic  solution  of  di- 
methyl-amido-azobenzol.  A  red  color  instantly  appears  if 
free  hydrochloric  acid  be  present.  Add  decinormal  sodium 
hydroxid  solution,  drop  by  drop  from  a  buret,  until  the  last 
trace  of  red  just  disappears,  and  a  canary-yellow  color 
takes  its  place  (Plate  XI,  C,  C')-  Read  off  the  number  of 
cubic  centimeters  of  decinormal  solution  added,  and  cal- 
culate the  degrees,  or  percentage  of  free  hydrochloric 
acid,  as  in  Topfer's  method  for  total  acidity. 

When  it  is  impossible  to  obtain  sufl&cient  fluid  for  all  the 


PLATE  XI 


k~ 


A,  Gastric  fluid  to  which  a  i  per  cent,  solution  of  phenol phthalein 
has  been  added;  B,  gastric  fluid  to  which  a  i  per  cent,  solution  of  alizarin 
has  been  added;  C,  gastric  fluid  to  which  a  0.5  per  cent,  solution  of 
dimethylamido-azobenzol  has  been  added;  A',  A  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  B',  B  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  C,  C  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid  (Boston). 


EXAMINATION    OF   THE    GASTRIC   CONTENTS         411 

tests,  it  will  be  found  convenient  to  estimate  the  free  hydro- 
chloric acid  and  total  acidity  in  the  same  portion,  and  this 
is  frequently  adopted  as  a  routine  regardless  of  the 
amount  of  fluid  available.  After  fmding  the  free  hydro- 
chloric acid  as  just  described,  add  4  drops  phenolphthalein 
solution,  and  continue  the  titration.  The  amount  of  deci- 
normal  solution  used  in  both  titrations  indicates  the  total 
acidity. 

Topfer's  Method  for  Combined  Hydrochloric  Acid. — In 
a  beaker  take  10  c.c.  filtered  gastric  juice  and  add  4  drops 
of  the  indicator,  a  i  per  cent,  aqueous  solution  of  sodium 
alizarin  sulphonate.  Titrate  with  decinormal  sodium  hy- 
droxid  until  the  appearance  of  a  bluish-violet  color  which 
does  not  become  deeper  upon  addition  of  another  drop 
(Plate  XI,  B,  B').  It  is  difficult,  without  practice,  to 
determine  when  the  right  color  has  been  reached.  A 
reddish  violet  appears  first.  The  shade  which  denotes  the 
end  reaction  can  be  produced  by  adding  2  or  3  drops  of  the 
indicator  to  5  c.c.  of  i  per  cent,  sodium  carbonate  solution. 

Calculate  the  number  of  cubic  centimeters  of  decinormal 
solution  which  would  be  required  for  100  c.c.  of  stomach 
fluid.  This  gives,  in  degrees,  all  the  acidity  except  the 
combined  hydrochloric  acid.  The  combined  hydrochloric 
acid  is  then  found  by  deducting  this  amount  from  the  total 
acidity,  which  has  been  previously  determined. 

Example. — Suppose  that  5  c.c.  of  decinormal  solution  were 
required  to  produce  the  purple  color  in  10  c.c.  gastric  juice; 
then  5  X  10  =  50  =  a//  the  acidity  except  combined  hydro- 
chloric acid.  Suppose,  now,  that  the  total  acidity  has  al- 
ready been  found  to  be  70  degrees;  then  70  —  50  =  20 
degrees  of  combined  hydrochloric  acid;  and  20  X  0.00365  = 
0.073  P^^  cent. 

When  free  hydrochloric  acid  is  absent,  it  is  probably 
more  helpful  to  estimate  the  acid  deficit  than  the  com- 


412  THE    STOMACH 

bined  hydrochloric  acid.  The  acid  deficit  shows  how 
far  the  acid  secreted  by  the  stomach  falls  short  of  satu- 
rating the  protein  (and  bases)  of  the  meal.  It  repre- 
sents the  amount  of  hydrochloric  acid  which  must  be 
added  to  the  fluid  before  a  test  for  free  hydrochloric 
acid  can  be  obtained.     It  is  determined  by  titrating 

with  —  hydrochloric   acid,   using    dimethyl-amido-azo- 

benzol  as  indicator,  until  the  fluid  assumes  a  red  color. 
The  amount  of  deficit  is  expressed  by  the  number  of 
cubic  centimeters  of  the  decinormal  solution  required 
for  lOO  c.c.  of  the  stomach  fluid. 

(3)  Organic  Acids. — There  is  no  simple  direct  quan- 
titative method.  After  the  total  acidity  has  been  deter- 
mined, organic  acids  may  be  removed  from  another 
portion  of  the  gastric  filtrate  by  shaking  thoroughly 
with  an  equal  volume  of  neutral  ether,  allowing  the 
fluids  to  separate,  and  repeating  this  process  until  the 
gastric  fluid  has  been  extracted  with  eight  or  ten  times 
its  volume  of  ether.  The  total  acidity  is  then  deter- 
mined, and  the  difi^erence  between  the  two  determina- 
tions indicates  the  amount  of  organic  acids. 

(4)  Pepsin. — No  direct  method  is  available.  The 
following  are  sufficient  for  clinical  purposes: 

I.  Hammerschlag's  Method.— To  the  white  of  an  egg  add 
twelve  times  its  volume  of  0.4  per  cent,  hydrochloric  acid 
(dilute  hydrochloric  acid,  U.  S.  P.,  4  c.c;  water,  96  c.c), 
mix  well,  and  filter.  This  gives  a  i  per  cent,  egg-albumen 
solution.  Take  10  c.c  of  this  solution  in  each  of  three  tubes 
or  beakers.  To  A  add  5  c.c.  gastric  juice;  to  B,  5  c.c.  water 
with  0.5  Gm.  pepsin;  to  C,  5  c.c.  water  only.  Place  in  an 
incubator  for  an  hour  and  then  determine  the  amount  of 


EXAMINATION    OF    THE    GASTRIC   CONTENTS        413 

albumin  in  each  mixture  by  Esbach's  method.  Tube  C 
shows  the  amount  of  albumin  in  the  test-solution.  The 
difference  between  C  and  B  indicates  the  amount  of  albumin 
which  would  be  digested  by  normal  gastric  juice.  The 
difference  between  C  and  A  gives  the  albumin  which  is 
digested  by  the  fluid  under  examination.  Schiitz  has 
shown  that  the  amounts  of  pepsin  in  two  fluids  are  pro- 
portionate to  the  squares  of  the  products  of  digestion. 
Thus,  if  the  amounts  of  albumin  digested  in  tubes  A  and  B 
are  to  each  other  as  2  is  to  4,  the  amounts  of  pepsin  are  to 
each  other  as  4  is  to  16. 

Certain  sources  of  error  can  be  eliminated  by  diluting  the 
gastric  juice  several  times  before  testing.  The  most  im- 
portant of  these  are  that  the  law  of  Schiitz  holds  good  only 
for  comparatively  dilute  solutions,  and  that  the  products 
of  peptic  activity  inhibit  digestion. 

2.  Mett's  method  is  generally  preferred  to  the  preceding. 
Put  three  or  four  Mett's  tubes  about  2  cm.  long  into  a  small 
beaker  with  diluted  gastric  juice  (i  c.c.  of  the  filtrate  plus 
15  c.c.  twentieth-normal  hydrochloric  acid).  Place  in  an 
incubator  for  twenty-four  hours,  and  then  measure  as 
accurately  as  possible  in  millimeters  the  column  which 
has  been  digested,  using  a  millimeter  scale  and  a  hand 
lens  or,  better,  a  low  power  of  the  microscope  and  an  eye- 
piece micrometer.  Square  the  average  length  of  this 
column  (law  of  Schiitz)  and  multiply  by  the  degree  of 
dilution,  16.  The  maximum  figure  obtained  in  this  way 
is  256,  representing  a  digested  column  of  4  mm. 

Prepare  Mett's  tubes  as  follows: 

Beat  up  slightly  the  whites  of  one  or  two  eggs  and  filter. 
Pour  into  a  wide  test-tube  and  stand  in  this  a  number  of 
capillary  glass  tubes,  i  to  2  mm.  in  diameter.  When  the 
tubes  are  filled,  plug  their  ends  with  bread  crumbs,  and 
coagulate  the  albumin  by  heating  in  water  just  short  of 
boiling.     Dip  the  ends  of  the  tubes  in  melted  paraffin  and 


414  '^HE    STOMACH 

preserve  until  needed.  Bubbles,  if  present,  will  probably 
disappear  in  a  few  days.  When  wanted  for  use,  cut  the 
tubes  into  lengths  of  about  2  cm.  Discard  any  in  which  the 
albumin  has  separated  from  the  wall. 

D.  Microscopic  Examination 

A  drop  of  unliltered  stomach-contents  is  placed  upon 
a  slide,  covered  with  a  cover-glass,  and  examined  with 
the  i6-mm.  and  4-mm.  objectives.  A  drop  of  Lugol's 
solution  allowed  to  run  under  the  cover  will  aid  in  dis- 
tinguishing the  various  structures.  As  a  rule,  the  mi- 
croscopic examination  is  of  little  value. 

Under  normal  conditions  little  is  to  be  seen  except 
great  numbers  of  starch-granules,  with  an  occasional 
epithelial  cell,  yeast-cell,  or  bacterium.  Starch-gran- 
ules are  recognized  by  their  concentric  striations  and  the 
fact  that  they  stain  blue  with  iodin  solutions  when  undi- 
gested, and  reddish,  due  to  erythrodextrin,  when  par- 
tially digested. 

Pathologically,  remnants  of  food  from  previous  meals, 
red  blood-corpuscles,  pus-cells,  sarcinaj,  and  excessive 
numbers  of  yeast-cells  and  bacteria  may  be  encountered 
(Fig.  148). 

Remnants  of  food  from  previous  meals  indicate 
deficient  gastric  motility. 

Red  Blood-corpuscles.^ — Blood  is  best  recognized  by 
the  chemic  tests  already  given.  The  corpuscles  some- 
times retain  a  fairly  normal  appearance,  but  are  gener- 
ally so  degenerated  that  only  granular  pigment  is  left. 
When  only  a  few  fresh-looking  corpuscles  are  present, 
they  usually  come  from  irritation  of  the  mucous  mem- 
brane by  the  tube. 


EXAMINATION    OF    THE    GASTRIC   CONTENTS        415 

Pus-cells.^ — Pus  is  rarely  encountered  in  the  fluid 
removed  after  a  test-meal.  Considerable  numbers  of 
pus-corpuscles  have  been  found  in  some  cases  of  gastric 
cancer.  The  corpuscles  are  usually  partially  digested, 
so  that  only  the  nuclei  are  seen.  Swallowed  sputum 
must  always  be  considered. 

.Sarcinae.^ — These  are  small  spheres  arranged  in  cuboid 
groups,  often  compared  to  bales  of  cotton.     They  fre- 


Fig.  148. — General  view  of  the  gastric  contents:  a,  Squamous  epi- 
thelial cells  from  esophagus  and  mouth;  b,  leukocytes;  c,  cylindric  epi- 
thelial cells;  d,  muscle-fibers;  e,  fat-droplets  and  fat-crystals;/,  starch- 
granules;  g,  chlorophyl-containing  vegetable  matters;  h,  vegetable 
spirals;  i,  bacteria;  k,  sarcinae;  I,  yeast  cells  (Jakob). 


quently  form  large  clumps  and  are  easily  recognized. 
They  stain  brown  with  iodin  solution.  They  signify  fer- 
mentation. Their  presence  is  evidence  against  the 
existence  of  gastric  cancer,  in  which  disease  they  rarely 
occur. 

Yeast-cells. — As  already  stated,  a  few  yeast-cells 
may  be  found  under  normal  conditions.  The  presence 
of  considerable  numbers  is  evidence  of  retention  and 


4i6 


THE    STOMACH 


fermentation.  Their  appearance  has  been  described 
(see  p.  239).     They  stain  brown  with  iodin  solution. 

Bacteria. — Numerous  bacteria  may  be  encountered, 
especially  in  the  absence  of  free  hydrochloric  acid.  The 
Boas-Oppler  bacillus  is  the  only  one  of  special  signifi- 
cance. It  occurs  in  the  majority  of  cases  of  cancer,  and 
is  rarely  found  in  other  conditions.  Carcinoma  probably 
furnishes  a  favorable  medium  for  its  growth. 

These  bacilli  (Fig.  149)  are  large  (5  to  10  /j.  long), 
non-motile,  and  usually  arranged  in  clumps  or  end  to 


Fig.  149. — Boas-Oppler  bacilli  from  case  of  gastric  cancer  (Boston). 

end  in  zig-zag  chains.  They  stain  brown  with  iodin 
solution,  which  distinguishes  them  from  Lepiothrix  buc- 
calis  (see  p.  535),  which  is  not  infrequently  swallowed, 
and  hence  found  in  stomach  fluid.  They  also  stain  by 
Gram's  method.  They  are  easily  seen  with  the  4-mm. 
objective  in  unstained  preparations,  but  are  best  recog- 
nized with  the  oil  lens,  after  drying  some  of  the  fluid 
upon  a  cover-glass,  fixing,  and  staining  with  a  simple 
bacterial  stain  or  by  Gram's  method. 

A  few  large  non-motile  bacilli  are  frequently  seen; 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        417 

they  cannot  be  called  Boas-Oppler  bacilli  unless 
they  are  numerous  and  show  something  of  the  typical 
arrangement. 

E.  The  Gastric  Contents  in  Disease 

In  the  diagnosis  of  stomach  disorders  the  practitioner 
must  be  cautioned  against  relying  too  much  upon  exami- 
nations of  the  stomach-contents.  A  first  examination 
is  especially  unreliable.  Even  when  repeated  examina- 
tions are  made,  the  laboratory  findings  must  never  be 
considered  apart  from  the  clinical  signs. 

The  more  characteristic  findings  in  certain  disorders 
are  suggested  here: 

1.  Dilatation  of  the  Stomach. — Evidences  of  re- 
tention and  fermentation  are  the  chief  characteristics 
of  this  condition.  Hydrochloric  acid  is  commonly 
diminished.  Pepsin  may  be  normal  or  slightly  dimin- 
ished. Lactic  acid  may  be  detected  in  small  amounts, 
but  is  usually  absent  when  the  stomach  has  been  washed 
before  giving  the  test-meal.  Both  motility  and  ab- 
sorptive power  are  deficient.  The  microscope  com- 
monly shows  sarcinffi,  bacteria,  and  great  numbers  of 
yeast-cells.  Remnants  of  food  from  previous  meals  can 
be  detected  with  the  naked  eye  or  microscopically. 

2.  Gastric  Neuroses. — The  findings  are  variable. 
Successive  examinations  may  show  normal,  increased, 
or  diminished  hydrochloric  acid,  or  even  entire  absence 
of  the  free  acid.     Pepsin  is  usually  normal. 

The  presence  of  more  than  100  c.c.  of  gastric  juice 
in  the  fasting  stomach  has  until  lately  been  taken  to 
indicate  a  neurosis  characterized  by  continuous  hyper- 
secretion   (gastrosuccorrhea),    but    recent   studies   of 

27 


41 8  THE    STOMACH 

the  fasting  contents  with  the  Rehfus  tube  throw 
some  doubt  upon  the  condition.  When  the  fluid  con- 
tains food-particles,  it  is  the  result  of  retention,  not 
hypersecretion. 

3.  Chronic  Gastritis. — Free  hydrochloric  acid  may 
be  increased  in  early  cases.  It  is  generally  diminished 
in  well-marked  cases,  and  is  often  absent  in  advanced 
cases.  Lactic  acid  is  often  present  in  traces,  rarely  in 
notable  amount.  Secretion  of  pepsin  and  rennin  is 
always  diminished  in  marked  cases.  Mucus  is  fre- 
quently present,  and  is  very  significant  of  the  disease. 
Motility  and  absorption  are  generally  deficient.  Small 
fragments  of  mucous  membrane  may  be  found,  and 
when  examined  by  a  pathologist,  may  occasionally 
establish  the  diagnosis. 

4.  Achylia  Gastrica  (Atrophic  Gastritis). — This 
condition  may  be  a  terminal  stage  of  chronic  gastritis. 
It  is  sometimes  associated  with  the  blood-picture  of 
pernicious  anemia.  It  gives  a  great  decrease,  and 
sometimes  entire  absence  of  hydrochloric  acid  and 
ferments.  The  total  acidity  may  be  as  low  as  i  or  2 
degrees.  Small  amounts  of  lactic  acid  may  be  present. 
Absorption  and  motility  are  not  greatly  affected. 

5.  Gastric  Carcinoma. — As  far  as  the  laboratory 
examination  goes,  the  cardinal  signs  are  absence  of  free 
hydrochloric  acid  and  presence  of  a  peptid-splitting 
ferment,  of  lactic  acid,  and  of  the  Boas-Oppler  bacillus. 
These  findings  are,  however,  by  no  means  constant. 

It  is  probable  that  some  substance  is  produced  by  the 
cancer  which  neutralizes  the  free  hydrochloric  acid, 
and  thus  causes  it  to  disappear  earlier  than  in  other 
organic  diseases  of  the  stomach.     The  peptid-splitting 


EXAMINATIONS  AS  TO  THE  CONDITION  OF  STOMACH  419 

ferment  (see  p.  405)  is  also  probably"  a  product  of  the 
cancer. 

The  presence  of  lactic  acid  is  possibly  the  most  sug- 
gestive single  symptom  of  gastric  cancer.  In  the  great 
majority  of  cases  its  presence  in  notable  amount  (o.i 
per  cent,  by  Strauss'  method)  after  Boas'  breakfast,  the 
stomach  having  been  washed  the  evening  before,  war- 
rants a  tentative  diagnosis  of  malignancy. 

Carcinoma  seems  to  furnish  an  especially  favorable 
medium  for  the  growth  of  the  Boas-Oppler  bacillus, 
hence  this  micro-organism  is  frequently  present. 

Blood  can  be  detected  in  the  stomach  fluid  by  the 
chemic  tests  in  nearly  one-half  of  the  cases,  and  is  more 
common  when  the  new  growth  is  situated  at  the  pylorus. 
Blood  is  present  in  the  stool  in  nearly  every  case. 

Evidences  of  retention  and  fermentation  are  the  rule 
in  pyloric  cancer.  Tumor  particles  are  sometimes 
found  late  in  the  disease. 

6.  Gastric  Ulcer.^ — There  is  excess  of  free  hydro- 
chloric acid  in  about  one-half  of  the  cases.  In  other 
cases  the  acid  is  normal  or  diminished.  Blood  is  often 
present.  The  diagnosis  must  be  based  largely  upon  the 
clinical  symptoms,  and  where  ulcer  is  strongly  suspected, 
it  is  probably  unwise  to  use  the  stomach-tube. 

II.  ADDITIONAL  EXAMINATIONS  WHICH  GIVE  INFOR- 
MATION AS  TO  THE  CONDITION  OF  THE  STOMACH 

1.  Absorptive  Power  of  the  Stomach. — This  is  a 
very  unimportant  function,  only  a  few  substances 
being  absorbed  in  the  stomach.  It  is  delayed  in  most 
organic  diseases  of  the  stomach,  especially  in  dilatation 
and  carcinoma,  but  not  in  neuroses.  The  test  has  little 
practical  value. 


420  THE   STOMACH 

Give  the  patient,  upon  an  empty  stomach,  a  3-gr.  cap- 
sule of  potassium  iodid  with  a  glass  of  water,  taking  care 
that  none  of  the  drug  adheres  to  the  outside  of  the  capsule. 
At  intervals  test  the  saliva  for  iodids  by  moistening  starch- 
paper  with  it  and  touching  with  yellow  nitric  acid.  A  blue 
color  shows  the  presence  of  an  iodid,  and  appears  normally 
in  ten  to  thirty  minutes  after  ingestion  of  the  capsule.  A 
longer  time  denotes  delayed  absorption. 

Starch-paper  is  prepared  by  soaking  filter-paper  in  boiled 
starch  and  drying. 

2.  Motor  Power  of  the  Stomach. — This  refers  to 
the  rapidity  with  which  the  stomach  passes  its  contents 
on  into  the  intestines.  It  is  very  important:  intestinal 
digestion  can  compensate  for  insufhcient  or  absent 
stomach  digestion  only  so  long  as  the  motor  power  is 
good. 

Motility  is  impaired  to  some  extent  in  chronic  gas- 
tritis. It  is  especially  deficient  in  dilatation  of  the 
stomach  due  to  atony  of  the  gastric  wall  or  to  pyloric 
obstruction,  either  benign  or  malignant.  It  is  increased 
in  most  conditions  with  hyperchlorhydria. 

The  best  evidence  of  deficient  motor  power  is  the 
detection  of  food  in  the  stomach  at  a  time  when  it 
should  be  empty,  e.g.^  before  breakfast  in  the  morning. 
A  special  test-meal  containing  easily  recognized  mate- 
rials {e.g.,  rice  pudding  with  currants)  is  sometimes  given 
and  removed  at  the  end  of  six  or  seven  hours.  When 
more  than  100  c.c.  of  fluid  are  obtained  with  the  tube  one 
hour  after  an  Ewald  breakfast,  deficient  motility  may 
be  inferred. 

Ewald's  salol  test  is  scarcely  so  reliable  as  the  above.  It 
depends  upon  the  fact  that  salol  is  not  absorbed  until  it 


EXAMINATIONS  AS  TO  THE  CONDITION  OP  STOMACH  42 1 

reaches  the  intestines  and  is  decomposed  by  the  alkaline 
intestinal  juices. 

The  patient  is  given  15  gr.  of  salol  with  a  test-breakfast, 
and  the  urine,  passed  at  intervals  thereafter,  is  tested  for 
salicyluric  acid.  A  few  drops  of  10  per  cent,  ferric  chlorid 
solution  are  added  to  a  small  quantity  of  the  urine..  A  violet 
color  denotes  the  presence  of  salicyluric  acid.  It  appears 
normally  in  sixty  to  seventy-five  minutes  after  ingestion  of 
the  salol.     A  longer  time  indicates  impaired  motor  power. 

3.  To  Determine  Size  and  Position  of  Stomach. 

— After  removing  the  test-meal,  while  the  tube  is  still  in 
place  force  quick  pufTs  of  air  into  the  stomach  by  com- 
pression of  the  bulb.  The  puffs  can  be  clearly  heard 
with  a  stethoscope  over  the  region  of  the  stomach,  and 
nowhere  else. 

4.  Sahli's  Desmoid  Test  of  Gastric  Digestion. — 
Two  pills,  one  containing  o.i  Gm.  iodoform,  the  other 
0.05  Gm.  methylene-blue,  are  wrapped  in  little  bags 
made  of  thin  sheets  of  rubber  and  tied  with  a  string  of 
raw  catgut.  No.  oo.  The  bags  must  be  carefully  folded 
and  tied.  Before  use  they  should  be  placed  for  a  time 
in  water.  If  they  float  or  if  any  of  the  methylene- 
blue  escapes  and  colors  the  water  they  are  useless  for 
the  test. 

The  patient  swallows  the  two  bags  with  the  aid  of  a 
little  water  during  the  noon  meal,  and  the  urine  is  tested 
at  intervals  thereafter.  According  to  Sahli,  the  catgut 
is  digested  by  gastric  juice  and  not  by  pancreatic  or 
intestinal  juices.  If  gastric  digestion  is  normal,  iodin 
and  methylene-blue  can  be  detected  in  the  urine  in  the 
afternoon  or  evening  of  the  same  day.  The  reaction 
may  occur  when  digestion  is  very  poor,  provided  gastric 


422  THE    STOMACH 

motility  is  diminished,  but  it  is  then  delayed.  If  the 
reaction  does  not  appear,  gastric  digestion  has  not 
occurred. 

Methylene-blue  is  recognized  in  the  urine  by  the  green  or 
blue  color  which  it  imparts.  It  is  sometimes  eliminated  as 
a  chromogen,  in  which  case  a  little  of  the  urine  must  be 
acidified  with  acetic  acid  and  boiled  to  bring  out  the  color. 

To  detect  the  iodin,  some  of  the  urine  is  decolorized  by 
gently  heating  and  filtering  through  animal  charcoal.  To 
ID  c.c.  are  then  added  i  c.c.  dilute  sulphuric  acid,  and  0.5 
c.c.  of  a  I  per  cent,  solution  of  sodium  nitrite  and  2  c.c.  of 
chloroform.  Upon  shaking,  a  rose  color  will  be  imparted  to 
the  chloroform  if  iodin  be  present.  Another  method  of 
testing  for  iodin  is  given  on  page  194. 


CHAPTER  V 
THE  FECES 

As  commonly  practised,  an  examination  of  the  feces  is 
limited  to  a  search  for  intestinal  parasites  or  their  ova. 
Much  of  value  can,  however,  be  learned  from  other 
simple  examinations,  particularly  a  careful  inspection. 
Anything  approaching  a  complete  analysis  is,  on  the 
other  hand,  a  waste  of  time  for  the  clinician. 

The  normal  stool  is  a  mixture  of — (a)  water;  (b) 
undigested  and  indigestible  remnants  of  food,  as  starch- 
granules,  particles  of  meat,  vegetable  cells  and  fibers, 
etc.;  (c)  digested  foods,  carried  out  before  absorption 
can  take  place;  (d)  products  of  the  digestive  tract,  as 
altered  bile-pigments,  enzymes,  mucus,  etc. ;  (e)  products 
of  decomposition,  as  indol,  skatol,  fatty  acids,  and  vari- 
ous gases;  (/)  epithelial  cells  shed  from  the  wall  of  the 
intestinal  canal ;  (g)  harmless  bacteria,  which  are  always 
present  in  enormous  numbers. 

Pathologically,  we  may  find  abnormal  amounts  of 
normal  constituents,  blood,  pathogenic  bacteria,  animal 
parasites  and  their  ova,  and  biliary  and  intestinal 
concretions. 

The  stool  to  be  examined  should  be  passed  into  a  clean 
vessel,  without  admixture  of  urine.  The  examination 
should  not  be  delayed  more  than  a  few  hours,  owing  to 
the  changes  caused  by  decomposition.     The  offensive 

423 


424  THE   PECES 

odor  can  be  partially  overcome  with  turpentine,  5  "per 
cent,  phenol,  or  a  little  formalin.  When  search  for 
amebae  is  to  be  made,  the  vessel  must  be  warm,  and  the 
stool  kept  warm  until  examined;  naturally,  no  disin- 
fectant can  be  used.  For  other  protozoa  a  saline 
cathartic  may  be  given  and  the  second  stool  examined. 
The  first  stool  is  usually  too  solid,  and  the  later  ones  too 
greatly  diluted. 

I.  MACROSCOPIC  EXAMINATION 

1.  Quantity. — The  amount  varies  greatly  with  diet 
and  other  factors.  The  average  is  about  100  to  150  Gm. 
in  twenty-four  hours.  It  is  much  larger  upon  a  vege- 
table diet. 

2.  Frequency. — One  or  two  stools  in  twenty-four 
hours  may  be  considered  normal,  yet  one  in  three  or  four 
days  is  not  uncommon  with  healthy  persons.  The 
individual  habit  should  be  considered  in  every  case. 

3.  Form  and  Consistence.^ — Soft,  mushy,  or  liquid 
stools  follow  cathartics  and  accompany  diarrhea.  Co- 
pious, purely  serous  discharges  without  fecal  matter 
are  significant  of  Asiatic  cholera,  although  sometimes 
observed  in  other  conditions.  Hard  stools  accompany 
constipation.  Rounded  scybalous  masses  are  common 
in  habitual  constipation,  and  indicate  atony  of  the  mus- 
cular coat  of  the  colon.  Flattened,  ribbon-like  stools  re- 
sult from  some  obstruction  in  the  rectum,  generally  a 
tumor  or  a  stricture  from  a  healed  ulcer,  most  commonly 
syphilitic.  When  bleeding  piles  are  absent,  blood- 
streaks  upon  such  a  stool  point  to  carcinoma. 

4.  Color. — The  normal  light  or  dark-brown  color  is 
due  chiefly  to  urobilin,  which  is  formed  from  bilirubin 


MACROSCOPIC   EXAMINATION  425 

by  reduction  processes  in  the  intestine,  largely  the 
result  of  bacterial  activity.  The  stools  of  infants  are 
yellow,  owing  partly  to  their  milk  diet  and  partly  to  the 
presence  of  unchanged  bilirubin. 

Diet  and  drugs  cause  marked  changes:  milk,  a  light 
yellow  color;  cocoa  and  chocolate,  dark  gray;  various 
fruits,  reddish  or  black;  iron  and  bismuth,  dark  brown 
or  black;  hematoxylin,  red,  etc. 

Pathologically,  the  color  is  important.  A  golden  yel- 
low is  generally  due  to  unchanged  bilirubin.  Green 
stools  are  not  uncommon,  especially  in  diarrheas  of  child- 
hood. They  are  sometimes  met  in  apparently  healthy 
infants,  alternating  with  normal  yellow  stools,  and  have 
little  significance  unless  accompanied  by  symptoms. 
The  color  is  due  to  biliverdin  or,  sometimes,  to  chromo- 
genic  bacteria.  Putty-colored  or  "acholic"  stools 
occur  when  bile  is  deficient,  either  from  obstruction  to 
outflow  or  from  deficient  secretion.  The  color  is  due 
less  to  absence  of  bile-pigments  than  to  presence  of  fat. 
Sir^ilar  stools,  which  manifestly  consist  largely  of  fat, 
are  fcommon  in  conditions  like  tuberculous  peritonitis, 
which  interfere  with  absorption  of  fats,  and  in  pan- 
creatic disease. 

Notable  amounts  of  blood  produce  tarry  black  stools 
when  the  source  of  the  hemorrhage  is  the  stomach  or 
upper  intestine,  and  a  dark  brown  to  bright  red  as  the 
source  is  nearer  the  rectum.  When  diarrhea  exists  the 
color  may  be  red,  even  if  the  source  of  the  blood  is  high 
up.  Red  streaks  of  blood  upon  the  outside  of  the  stool 
are  due  to  lesions  of  rectum  or  anus. 

5.  Odor.— Products  of  decomposition,  chiefly  indol 
and  skatol,  are  responsible  for  the  normal  offensive  odor. 


426  THE    FECES 

The  strength  of  this  odor  depends  largely  upon  the 
amount  of  meat  in  the  diet  and  the  activity  of  putre- 
factive bacteria  in  the  intestine.  Upon  a  vegetable 
or  milk  diet  the  odor  is  much  less.  A  sour  odor  due 
to  fatty  acids  is  normal  for  nursing  infants,  and  is  noted 
in  mild  diarrheas  of  older  children.  In  the  severe 
diarrheas  of  childhood  a  putrid  odor  is  common.  In 
adults,  stools  emitting  a  very  foul  stench  are  suggestive 
of  malignant  or  syphilitic  ulceration  of  the  rectum  or 
gangrenous  dysentery. 

6.  Mucus. — Excessive  quantities  of  mucus  are  easily 
detected  with  the  naked  eye,  and  signify  irritation  or 
inflammation.  When  the  mucus  is  small  in  amount  and 
intimately  mixed  with  the  stool,  the  trouble  is  probably 
in  the  small  intestine.  Larger  amounts,  not  well  mixed 
with  fecal  matter,  indicate  inflammation  of  the  large 
intestine.  Stools  composed  almost  wholly  of  mucus 
and  streaked  with  blood  are  the  rule  in  dysentery, 
ileocolitis,  and  intussusception. 

In  the  so-called  mucous  colic  or  membranous  enteritis, 
shreds  and  ribbons  of  altered  mucus,  sometimes  repre- 
senting complete  casts  of  portions  of  the  bowel,  are 
passed.  These  may  appear  as  firm,  irregularly  seg- 
mented strands,  suggesting  tapeworms.  The  mucus 
sometimes  takes  the  form  of  frog-spawn-like  masses. 
In  some  cases  it  is  passed  at  variable  intervals,  with 
colic;  in  others,  with  every  stool,  with  only  vague  pains 
and  discomfort.  It  is  distinguished  from  inflammatory 
mucus  by  absence  of  pus-corpuscles.  The  condition  is 
not  uncommon  and  should  be  more  frequently  recog- 
nized. It  is  probably  a  secretory  neurosis,  hence  the 
name  "membranous  enteritis"  is  inappropriate. 


MACROSCOPIC    EXAMINATION  427 

7.  Concretions. — Gall-stones  should  be  searched 
for  in  every  case  of  obscure  colicky  abdominal  pain. 
Intestinal  concretions  (enteroliths)  are  rare.  Intestinal 
sand,  consisting  of  sand-like  grains,  is  common  in  neu- 
rotic conditions,  such  as  mucous  colitis.  After  inges- 
tion of  considerable  amounts  of  olive  oil,  nodules  of 
soap  and  fat  often  appear  in  the  feces,  and  may  be  mis- 
taken by  the  patient  for  gall-stones,  particularly  when 
the  oil  has  been  given  for  cholelithiasis. 

Concretions  can  be  found  by  breaking  up  the  fecal 
matter  in  a  sieve  (which  may  be  improvised  from  gauze) 
while  pouring  water  over  it.  It  must  be  remembered 
that  gall-stones,  if  soft,  may  go  to  pieces  in  the  bowel. 
Gall-stones  are  readily  identified  by  their  faceted  sur- 
faces. When  facets  are  absent  the  stones  can  be  dis- 
tinguished from  other  concretions  by  detecting  choles- 
terol and  bile-pigment  in  them.  The  stone  is  broken 
up  and  as  far  as  possible  dissolved  in  ether.  If  now  the 
ether  be  slowly  evaporated  in  a  watch-glass,  crystals  of 
cholesterol  (Fig.  49)  will  separate  out.  To  extract  bile- 
pigments  treat  the  parts  of  the  stone  which  have  failed 
to  dissolve  in  ether  with  chloroform  and  then  with 
hot  alcohol.  A  yellow  color  in  the  chloroform  and  a 
green  in  the  alcohol  show  the  presence  of  bilirubin 
and  biliverdin  respectively. 

8.  Animal  Parasites. — Segments  of  tapeworms  and 
the  adults  of  other  parasites  are  often  found  in  the  stool. 
The  smaller  ones  are  sought  as  described  for  concretions, 
the  material  caught  by  the  sieve  being  floated  out  in 
clear  water  and  examined  in  a  glass  dish  over  a  dark 
back  ground  placed  some  distance  below.  The  search 
should  be  preceded  by  a  vermicide  and  a  brisk  purge. 


428 


THE    FECES 


Patients  often  mistake  vegetable  tissue  for  intestinal 
parasites,  and  the  writer  has  many  times  known  phy- 
sicians to  make  similar  mistakes.  The  most  frequent 
sources  of  confusion  are  long  fibers  from  poorly  masti- 
cated celery  or  "greens,"  which  suggest  round  worms; 
cells  from  orange,  which  suggest  seatworms;  and  fibers 
from  banana,  which,  because  of  the  segmented  structure 
and  the  presence  of  oval  cells,  suggest  tapeworms  and 
ova  (Fig.  150).     Even  slight  familiarity  with  the  micro- 


FiG.  150. —  Undigested  fiber  from  center  of  banana,  in  feces  (  X  15). 
In  the  lower  part  of  the  figure  the  fiber  is  shown  natural  size.  The 
segments  are  colored  reddish-brown  when  found  in  the  stool.  Such 
fibers  are  often  reported  as  small  tapeworms. 

scopic  structure  of  vegetable   tissue  will  prevent  the 
chagrin  of  such  errors. 

9.  Curds. — The  stools  of  nursing  infants  frequently 
contain  whitish  curd-like  masses,  due  either  to  imper- 
fect digestion  of  fat  or  casein  or  to  excess  of  these  in  the 
diet.  When  composed  of  fat,  the  masses  are  soluble 
in  ether,  and  give  the  Sudan  III  test.  If  composed  of 
casein,  they  will  become  tough  and  fibrous-like  when 
placed  in  formalin  (lo  per  cent.)  for  twenty-four  hours. 


CHEMIC   EXAMINATION  429 

II.  CHEMIC  EXAMINATION 

Complicated  chemic  examinations  are  of  little  value 
to  the  clinician.     Certain  tests  are,  however,  important. 

I .  Blood. — When  present  in  large  amount  blood  pro- 
duces such  changes  in  the  appearance  of  the  stool  that 
it  is  not  likely  to  be  overlooked.  Traces  of  blood  (occult 
hemorrhage)  can  be  detected  only  by  special  tests. 
Recognition  of  occult  hemorrhage  has  its  greatest  value 
in  diagnosis  of  gastric  cancer  and  ulcer.  It  is  constantly 
present  in  practically  every  case  of  gastric  cancer,  and  is 
always  present,  although  usually  intermittently,  in  ulcer. 
Traces  of  blood  also  accompany  malignant  disease  of  the 
bowel,  the  presence  of  certain  intestinal  parasites,  and 
other  conditions. 

Detection  of  Occult  Hemorrhage. — Soften  a  portion  of  the 
stool  with  water,  shake  with  an  equal  volume  of  ether  to 
remove  fat,  and  discard  the  ether.  Treat  10  c.c.  of  the 
remaining  material  with  about  one-third  its  volume  of 
glacial  acetic  acid  and  extract  with  about  10  c.c.  ether. 
Should  the  ether  not  separate  well,  add  a  Uttle  alcohol 
and  mix  gently.  Apply  the  guaiac  or  benzidin  test  to  the 
ether  as  already  described  (see  p.  181).  This  will  require 
only  a  portion  of  the  ether-extract.  In  case  the  test  is 
positive,  it  is  a  good  plan  to  use  the  remainder  for  spec- 
troscopic examination  treating  it  so  as  to  produce  the 
bands  of  hemochromogen  (see  pp.  369,  371). 

Wagner  makes  a  thick  smear  of  the  feces  on  a  glass  slide 
by  means  of  a  wooden  spatula,  allows  this  to  dry,  and  pour 
the  mixed  benzidin  reagent  on  it.  The  blue  color  is  recog- 
nized macroscopically  and  microscopically. 

In  every  case  iron-containing  medicines  must  be  stopped, 
and  blood-pigment  must  be  excluded  from  the  food  by  giving 


430  THE   FECES 

an  appropriate  diet,  e.g.,  bread,  milk,  eggs,  and  fruit.  At 
the  beginning  of  the  restricted  diet  give  a  gram  of  powdered 
charcoal  or,  better,  0.3  Gm.  of  carmin,  in  capsules,  so  as  to 
mark  the  corresponding  stool. 

2.  Bile. — Normally,  unaltered  bile-pigment  is  never 
present  in  the  feces  of  adults.  In  catarrhal  conditions 
of  the  small  intestine  bilirubin  may  be  carried  through 
unchanged.  It  may  be  demonstrated  by  the  Schmidt 
test  for  urobilin  which  follows,  or,  if  a  considerable 
amount  is  present,  by  filtering  (after  mixing  with  water 
if  the  stool  be  solid)  and  testing  the  filtrate  by  Gmelin's 
method,  as  described  under  The  Urine. 

3.  Urobilin  (Hydrobilirubin). — The  urobilin  of  the 
urine  and  the  hydrobilirubin  which  constitutes  the 
principal  normal  pigment  of  the  feces  appear  to  be 
identical;  and  the  present  tendency  is  to  use  the  name 
"urobilin"  in  both  cases.  In  a  general  way,  the  name 
covers  both  the  pigment,  urobilin,  and  the  chromogen, 
urobilinogen,  of  which  it  is  an  oxidation  product,  since 
the  two  substances  have  exactly  the  same  significance. 
For  the  mode  of  formation  and  the  significance  in  the 
urine  the  reader  is  referred  to  the  chapter  on  the  urine. 
Owing  to  constipation  and  other  factors,  the  amount 
of  urobilin  in  the  feces  is  subject  to  marked  daily  varia- 
tions. The  average  of  a  number  of  successive  daily 
estimations  is,  however,  fairly  constant  Ordinarily  the 
twenty-four-hour  stool  gives  a  dilution  value  (see 
method  below)  of  6000;  and  9000  may  be  taken  as  the 
upper  normal  limit. 

Since  bilirubin,  its  mother  substance,  is  a  product  of 
blood-pigment,  an  abnormally  large  amount  of  uro- 
bilin in  the  feces  may  be  taken  as  definite  evidence  of 


CHEMIC    EXAMINATION  43 1 

excessive  destruction  of  red  blood  cells  within  the 
circulation;  and  quantitative  estimations  are  of  great 
value  whenever  such  increased  blood  destruction  is  in 
question.  They  have  been  found  especially  useful 
in  distinguishing  the  anemias  due  to  excessive  hemolysis 
(e.g.,  pernicious  anemia)  from  other  anemias  in  which 
hemolysis  is  not  a  prominent  factor  (carcinoma,  hemor- 
rhage); in  following  the  progress  of  individual  cases 
of  pernicious  anemia;  and  in  studying  the.  effect  of 
splenectomy  performed  as  a  therapeutic  measure  in  this 
disease.  In  progressing  cases  of  pernicious  anemia  the 
Wilber  and  Addis  method  usually  gives  urobilin  dilution 
values  of  20,000  to  30,000  and  often  much  more. 
Urobilin  is  nearly  or  quite  absent  from  the  stool  in 
cases  of  obstruction  to  the  common  or  hepatic  bile- 
ducts. 

Detection.— The  chemical  tests  mentioned  on  p.  186 
may  be  applied  to  a  watery  extract  of  the  stool.  Di- 
rect spectroscopic  examination  is  impossible  owing  to 
the  cloudiness  of  the  suspension.  The  following  test  is 
also  useful: 

Schmidt's  Test. — Rub  up  a  small  quantity  of  the  fecal 
matter  with  saturated  mercuric  chlorid  solution  and  let 
stand  twenty-four  hours.  Urobilin  will  give  a  red  color, 
which  is  likewise  imparted  to  such  microscopic  structures 
as  are  stained  with  urobilin.  A  green  color  shows  the 
presence  of  unchanged  bilirubin  and  is  not  seen  normally. 

Quantitative  Estimation. — The  method  of  Wilber 
and  Addis  is  probably  most  useful  clinically.  While 
it  does  not  give  the  actual  quantity  of  urobilin,  it 
furnishes  a  rough  comparative  method  which  works 
very  well  in  practice.     Because  of  the  instability  of 


432  THE   FECES 

urobilin,  methods  which  involve  elaborate  treatment 
of  the  feces  are  not  applicable.  Since  urobilin  and 
urobilinogen  have  the  same  significance  and  are  so 
readily  changed  one  into  the  other  they  are  included 
together  in  the  estimation.  Estimations  are  valueless 
unless  the  average  of  six  to  ten,  made  on  successive 
days,  is  taken. 

Method  of  Wilber  and  Addis. — i.  Collect  all  the  feces 
for  twenty-four  hours,  keeping  them  in  darkness. 

2.  Grind  the  whole  quantity  with  water  to  a  homogeneous 
paste. 

3.  Dilute  to  1000  c.c.  with  tap  water  (or  to  500  c.c.  or 
2000  c.c.  if  the  amount  of  feces  is  unusually  small  or  large). 

4.  Measure  off  25  c.c.  and  add  to  this  75  c.c.  acid  alcohol 
(alcohol  64  c.c,  concentrated  hydrochloric  acid  i  c.c, 
water  32  c.c). 

5.  Place  in  a  mechanical  shaker  for  one-half  hour. 
Constant  shaking  by  hand  for  a  similar  period  will  answer. 

6.  Add  100  c.c.  of  saturated  alcoholic  solution  of  zinc 
acetate  and  filter. 

7.  To  20  c.c.  of  the  filtrate  add  2  c.c  of  Ehrlich's  reagent 
(para-dimethylamidobenzaldehyde,  20  Gm. ;  concentrated 
hydrochloric  acid,  150  c.c;  water,  150  c.c). 

8.  Keep  in  darkness  until  next  day  (or  at  least  for  six 
hours)  and  examine  spectroscopically.  In  the  presence  of 
both  urobilinogen  and  urobilin  the  absorption  bands 
indicated  in  Fig.  151,  A  and  B,  will  be  seen. 

9.  Dilute  with  60  per  cent,  alcohol,  adding  a  few  cubic 
centimeters  at  a  time,  until  first  one  and  then  the  other 
band  has  entirely  disappeared  when  the  slit  of  the  spectro- 
scope is  wide  open  but  still  remains  visible  when  the  slit 
is  partly  closed.  The  end-point  is  fairly  definite  after  one 
has  established  his  standard  upon  a  series  of  normal  stools. 
For  the  sake  of  uniformity  the  examination  may  be  made 


CHEMIC    EXAMINATION 


433 


in  a  50-c.c.  cylinder  graduate,  in  a  dark  room  by  the  light 
of  a  Mazda  electric  bulb,  with  the  spectroscope  held  close 
to  the  light. 

10,  Calculate  separately  the  number  of  dilutions  neces- 
sary to  cause  disappearance  of  each  of  the  absorption  bands 
and  add  the  two  together.  The  calculation  is  based  not 
upon  the  20  c.c.  of  filtrate  used,  but  upon  the  2)'^  c.c.  of 
fecal  suspension  represented  by  the  filtrate.  The  dilution 
value  for  the  twenty-four-hour  stool    (1000  c.c.  of  fecal 


aJ 

7Z 

0 

z: 

UJ 

UJ 

i-i 

< 

-J 

tu 

ri 

LU 

£^ 

UJ 

DC 

—J 

ce 

0 
* 

>- 

0 

A 

CO 

B  C 


Eb 


L 


A 


B 


Fig.  151. — Absorption  spectra  of  A,  urobilinogen  in  acid  solu- 
tion with  Ehrlich's  reagent  and  B,  urobilin  in  acid  solution  with  zinc 
acetate. 


suspension)  is  then  found  by  multiplying  this  figure  by 
400.  When  the  fecal  suspension  was  made  up  to  500  c.c. 
or  2000  c.c.  the  multiplier  would  of  course  be  200  or  800. 
This  final  result  indicates  the  number  of  dilutions  which 
would  be  necessary  if  all  the  urobihn  and  urobilinogen  of 
the  twenty-four-hour  stool  were  concentrated  in  the  2)-^  c.c. 
of  fecal  suspension  examined. 

Example. — Suppose  that  in  step  9  the  urobilinogen 
band  disappeared  when  the  20  c.c.  of  filtrate  had  been 
diluted  to  25  c.c.  and  the  urobilin  band  when  the  volume 

28 


434  THE   FECES 

reached  30  c.c,  then  the  dilution  values  for  the  2^2  c.c.  of 
feces  would  be  10  and  12  respectively  and  the  combined 
value  10+12  =  22.  The  total  dilution  value  of  the 
twenty-four-hour  stool  would  then  be  22  X  400  =  8800. 

4.  Pancreatic  Ferments. — Two  of  the  ferments  of 
the  pancreatic  juice — amylase  and  tr\-psin — are  nor- 
mally present  in  the  feces.  Lipase  can  usually  not  be 
detected.  In  pancreatic  disease  and  in  simple  obstruc- 
tion of  the  pancreatic  duct  these  ferments  are  diminished 
or  absent.  Quantitative  estimations  therefore  furnish 
a  valuable  aid  in  the  diagnosis  of  pancreatic  disease, 
particularly  when  carried  out  in  conjunction  with  an 
estimation  of  amylase  in  the  urine.  Results,  although 
less  reliable,  have  much  the  same  significance  as  those 
given  by  examination  of  the  duodenal  contents  removed 
through  the  duodenal  tube— a  procedure  to  which  the 
practitioner  will  hesitate  to  resort  owing  to  its  technical 
difficulties  and  the  discomfort  to  the  patient. 

Owing  to  constipation,  diet,  and  other  factors  there 
are  considerable  variations  in  the  amounts  of  ferments. 
It  is  therefore  essential  that  a  uniform  technic  be 
adopted.  The  following  directions  are  based  upon  the 
method  recommended  by  T.  R.  Brown  for  amylase. 
It  is  best  in  every  case  to  estimate  both  amylase  and 
trypsin,  but  if  the  examination  is  limited  to  one  fer- 
ment amylase  should  be  chosen,  since  the  action  of 
trypsin  may  be  simulated  by  erepsin  and  the  proteo- 
lytic activity  of  bacteria. 

Estimation  of  Pancreatic  Ferments  in  Feces. — i.  Upon 

the  evening  before  the  test,  limit  the  patient  to  a  Hght 
supper  and  give  a  high  enema  at  bed  time. 


CHEMIC    EXAMINATION  435 

2.  At  7:00  next  morning,  give  750  c.c.  (25  ounces)  of 
milk. 

3.  At  7:30  give  }'-2  ounce  of  Epsom  salts:  repeat  at  8:00. 

4.  At  8:30  give  a  glass  of  water  containing  I4  teaspoonful 
of  sodium  bicarbonate. 

5.  Save  all  the  feces  passed  up  to  2  p.m.  in  a  vesse^  con- 
taining 2  ounces  of  toluol.  Keep  in  a  cool  place.  If  less 
dian  400  c.c.  are  obtained  give  an  enema  of  i  pint  of 
water. 

6.  Dilute  the  whole  volume  of  feces  to  3000  c.c.  with 
normal  salt  solution,  mix  well  and  centrifugalize  a  portion 
for  five  minutes.  Use  the  supernatant  fluid  for  the  follow- 
ing tests: 

Estimation  of  Amylase. — i.  Prepare  a  i  per  cent.  soLxt- 
tion  of  soluble  starch  as  follows:  To  100  c.c.  cold  distiUed 
water  add  i  Gm.  soluble  starch  (Kahlbaum's  recommended) 
and  heat  gently  with  constant  stirring  until  dear. 

2.  Place  2  c.c.  of  this  solution  in  each  of  13  test-tubes. 

3.  To  these  tubes  add  the  supernatant  fluid  from  the 
centrifugalized  feces  as  follows: 

To  tube  I  add  1.8  c-c-  To  tube    8  add  0.4      cx- 

To  tube  2  add  i  .6  ex.  To  tube    9  add  0.2      ex. 

To  tube  3  add  i  .4  cc  To  tube  10  add  o.i      ex. 

To  tube  4  add  1 . 2  ex.  '  To  tube  11  add  0.05    ex. 

To  tube  5  add  i  .0  c.c  To  tube  12  add  0.025  c-C- 

To  tube  6  add  o .  S  c.c.  To  tube  13  add  none  (controD 
To  tube  7  add  o .  6  ex. 

Bring  the  quantity  in  each  tube  up  to  4  c.c.  with  normal 
salt  solution. 

4.  Place  the  tubes  in  an  incubator  or  water  bath  at 
about  38°C.^  for  one-half  hour. 

^  Variations  in  reaction  and  variatiiHis  in  temperature  from  37°  to 
4o°C.  exert  no  af^reciable  effect  upon  the  result. 


436  THE    FECES 

5.  Fill  all  tubes  with  tap  water  and  add  a  drop  of  weak 
iodin  solution  to  each.     Gram's  iodin  solution  will  answer, 

6.  If  amylase  be  present,  the  series  of  tubes  will  vary 
from  yellow  through  red-purple  to  pure  blue,  depending 
upon  complete  or  partial  digestion  of  the  starch.  The 
tube  before  the  one  in  which  the  first  definite  trace  of 
blue  appears  is  taken  as  the  measure  of  digestion.  Brown 
found  the  lowest  normal  to  be  the  tenth  tube,  corresponding 
to  60,000  units.  ^ 

Test  for  Trypsin. — The  well-known  Gross  test  may  be 
appUed  as  follows: 

1.  Prepare  a  i  :  1000  solution  of  casein  as  follows: 

Casein  (Gruebler's  preferred)  o.i  Gm.; 

Sodium  bicarbonate  o .  i  Gm. ; 

Distilled  water  100  c.c. 

Boil  for  one  minute,  stirring  constantly,  and  cool. 

2.  Place  5  c.c.  of  the  casein  solution  in  each  of  13  test- 
tubes  and  add  to  these  tubes  the  same  amounts  of  the 
fecal  suspension  as  were  used  for  the  amylase  test. 

3.  Place  the  tubes  in  the  incubator  or  a  water  bath  at 
38°C.  for  one  hour. 

4.  Test  for  digestion  of  casein  by  adding  a  few  drops  of 
3  per  cent,  acetic  acid  to  e^ch  tube.  Digestion  is  com- 
plete in  those  tubes  in  which  no  white  precipitate  forms. 

m.  MICROSCOPIC  EXAMINATION 

Care  must  be  exercised  in  selection  of  portions  for 
examination.  A  random  search  will  often  reveal 
nothing  of  interest.  A  small  bit  of  the  stool,  or  any 
suspicious-looking   particle,    is    placed    upon    a    slide, 

^  This  means  the  number  of  cubic  centimeters  of  i  per  cent,  starch 
solution  which  would  be  digested  by  the  3000  c.c.  of  fecal  suspension 
under  the  stated  conditions  of  time  and  temperature. 


MICROSCOPIC  EXAMINATION 


437 


thinned  with  water  if  necessary,  and  covered  with  a 
cover-glass.  As  emphasized  by  Bass  and  Johns  the 
layer  should  be  just  thin  enough  to  read  news-print 
through  it.  A  large  slide — about  2  by  3  inches^ — with 
a  correspondingly  large  cover  will  be  found  convenient. 
Most  of  the  structures  which  it  is  desired  to  see  can 
be  found  with  a  i6-mm.  objective.  Details  of  struc- 
ture must  be  studied  with  a  higher  power. 


Fig.  152. — Microscopic  elements  of  normal  feces:  a.  Muscle- fibers; 
h,  connective  tissue;  c,  epithelial  cells;  d,  white  blood-corpuscles;  e, 
spiral  vessels  of  plants;  f-h,  vegetable  cells;  i.  plant  hairs;  k,  triple 
phosphate  crystals;  I,  stone  cells.  Scattered  among  these  elements 
are  micro-organisms  and  debris  (after  v.  Jaksch). 

The  bulk  of  the  stool  consists  of  granular  debris. 
Among  the  recognizable  structures  (Fig.  152)  met  in 
normal  and  pathologic  conditions  are:  Remnants  of 
food,  epithelial  cells,  pus-corpuscles,  red  blood-cor- 
puscles, crystals,  bacteria,  and  ova  of  animal  parasites. 

1.  Remnants  of  Food. — These  include  a  great  va- 
riety of  structures  which  are  very  confusing  to  the 
student.  Considerable  study  of  normal  feces  is 
necessary  for  their  recognition. 


438  THE   FECES 

Vegetable  fibers  are  generally  recognized  from  their 
spiral  structure  or  their  pits,  dots,  or  reticulate  mark- 
ings ;  vegetable  cells,  from  their  double  contour  and  the 
chlorophyl  bodies  which  many  of  them  contain.  These 
cells  are  apt  to  be  mistaken  for  the  ova  of  parasites. 
Vegetable  hairs  (Fig.  153)  frequently  look  much  Hke 
the  larvae  of  some  of  the  worms.  Anything  like  a 
careful  examination  will,  however,  easily  distinguish 
them,  because  of  the  homogeneous  and  highly  re- 
fractile  wall,  the  distinct  central  canal  which  extends 


sV 


r 
t. 


Fig.   153. — Vegetable  hair  (.down  from  skin  of  peach)  in  feces  (  X  150). 
Compare  with  Fig.  193. 

the  whole  length,  and,  especially,  the  absence  of 
motion.  Starch-granules  sometimes  retain  their  orig- 
inal form,  but  are  ordinarily  not  to  be  recognized 
except  by  their  staining  reaction.  Potato  remains 
appear  in  colorless  translucent  masses  somewhat 
like  sago  grains  or  flakes  of  mucus.  Starch  strikes 
a  blue  color  with  Lugol's  solution  when  undigested; 
a  red  color,  when  slightly  digested.  Muscle-fibers 
are  yellow,  and  when  poorly  digested  appear  as  short, 
transversely   striated   cylinders   with   rather   squarely 


MICROSCOPIC   EXAMINATION  439 

broken  ends  (Fig.  154).  Generally,  the  ends  are 
rounded  and  the  stria tions  faint;  or  only  irregularly 
round  or  oval  yellow  masses  which  bear  little  re- 
semblance to  normal  muscle-tissue  are  found.  If  a 
little  eosin  solution  be  run  under  the  cover,  muscle- 
fibers  will  take  up  the  red  color  and  stand  out 
distinctly. 

Fats  occur  in  three  modifications:  neutral  fats,  fatty 
acids,  and  soaps.     Neutral  fats  are  present  in  very  small 


Fig.   154. — Poorly   digested   muscle-fiber   in    feces  showing  striations 

(X200). 

amounts  or  not  at  all  on  an  ordinary  diet.  They 
appear  as  droplets  or  yellowish  flakes,  depending 
upon  the  melting  point.  They  stain  strongly  with 
Sudan  III;  and  do  not  stain  with  dilute  carbol-fuchsin 
as  do  fatty  acids  and  soaps.  Fatty  acids  take  the 
form  of  flakes  like  those  of  neutral  fat,  or  of  needle- 
like crystals  which  are  generally  aggregated  into  thick 
balls    or    irregular    masses    in    which    the    individual 


44©  THE   FECES 

crystals  are  difficult  to  make  out.  When  treated 
with  Sudan  III  the  amorphous  flakes  take  a  Hghter 
orange  than  do  the  neutral  fats,  while  the  crystals 
do  not  stain.  Soaps — chiefly  calcium  soap — appear 
partly  as  well-defined  yellow  amorphous  flakes  or 
rounded  masses  suggesting  eggs,  partly  as  coarse  crystals. 
They  do  not  stain  with  Sudan  III  and  do  not  melt  when 
warmed  as  do  the  fatty  acids.  Connective  tissue  con- 
sists of  colorless  or  yellowish  threads  with  poorly  defined 
edges  and  indefinite  longitudinal  striations.  When 
treated  with  30  per  cent,  acetic  acid  the  fibers  swell  up 
and  become  clear  and  homogeneous.  Elastic  fibers, 
which  are  often  present  along  with  the  connective  tissue, 
are  more  definite  in  outline  and  branch  and  anastomose. 
They  are  rendered  more  distinct  by  acetic  acid. 

Excess  of  any  of  these  structures  may  result  from 
excessive  ingestion  or  deficient  digestion. 

2.  Epithelial  Cells. — A  few  cells  derived  from  the 
wall  of  the  alimentary  canal  are  a  constant  finding. 
They  show  all  stages  of  disintegration  and  are  often 
unrecognizable.  A  marked  excess  has  its  origin  in  a 
catarrhal  condition  of  some  part  of  the  bowel.  Squa- 
mous cells  come  from  the  anal  orifice;  otherwise  the 
form  of  the  cells  gives  no  clue  to  the  location  of  the 
lesion. 

3.  Pus. — Amounts  of  pus  sufficient  to  be  recognized 
with  the  eye  alone  indicate  rupture  of  an  abscess  into 
the  bowel.  If  well  mixed  with  the  stool,  the  source  is 
high  up,  but  in  such  cases  the  pus  is  apt  to  be  more 
or  less  completely  digested,  and  hence  unrecognizable. 
Small  amounts,  detected  only  by  the  microscope,  are 
present  in  catarrhal  and  ulcerative  conditions  of  the  in- 


MICROSCOPIC   EXAMINATION  44 1 

testine,  the  number  of  pus-cells  corresponding  to  the 
severity  and  extent  of  the  process. 

4.  Blood=corpuscles. — Unaltered  red  corpuscles  are 
rarely  found  unless  their  source  is  near  the  anus.  Ordi- 
narily, only  masses  of  blood-pigment  can  be  seen. 
Blood  is  best  recognized  by  the  chemic  tests  (see 
p.  429). 

5.  Bacteria. — In  health,  bacteria — mostly  dead — 
constitute  about  one-third  of  the  weight  of  the  dried 
stool.  They  are  beneficial  to  the  organism,  although 
not  actually  necessary  to  its  existence.  Under  certain 
conditions  they  may  be  harmful.  It  is  both-  difficult 
and  unprofitable  to  identify  them.  The  great  majority 
belong  to  the  colon  bacillus  group,  and  are  negative  to 
Gram's  method  of  staining. 

In  some  pathologic  conditions  the  character  of  the 
intestinal  flora  changes,  so  that  Gram-staining  bacteria 
very  greatly  predominate.  As  shown  by  R.  Schmidt, 
of  Neusser's  clinic  in  Vienna,  this  change  is  most  con- 
stant and  most  striking  in  cancer  of  the  stomach,  owing 
to  large  numbers  of  Boas-Oppler  bacilli,  and  is  of  some 
value  in  diagnosis.  He  believes  that  a  diagnosis  of 
gastric  carcinoma  should  be  very  unwillingly  made 
with  an  exclusively  "Gram-negative"  stool,  while  a 
"Gram-positive"  stool,  due  to  bacilli  (which  should 
also  stain  brown  with  Lugol's  solution),  may  be  taken 
as  very  strong  evidence  of  cancer.  A  Gram-positive 
stool  due  to  cocci  is  suggestive  of  intestinal  ulceration. 
The  technic  is  the  same  as  when  Gram's  method  is  appHed 
to  other  material  (see  p.  572),  except  that  the  smear  is 
fixed  by  immersion  in  methyl-alcohol  for  five  minutes 
instead  of  by  heat.     Pyronin  is  a  good  counterstain. 


442  THE    FECES 

The  deep  purple  Gram-staining  bacteria  stand  out  more 
prominently  than  the  pale-red  Gram-negative  organisms, 
and  one  maybe  misled  into  thinking  them  more  numerous 
even  in  cases  in  which  they  are  much  in  the  minority. 
The  number  of  Boas-Oppler  bacilli  can  be  increased  by 
administering  a  few  ounces  of  sugar  of  milk  the  day 
before  the  examination.  The  bacteria  can  be  obtained 
comparatively  free  from  food  remnants  by  mixing  a 
little  of  the  feces  with  water,  allowing  to  settle  for  a 
short  time,  and  making  smears  from  the  supernatant 
fluid.  One  must  of  course  be  on  his  guard  against 
Bacillus  •  bulgaricus  taken  with  artificial  buttermilk. 

Owing  to  the  difficulty  of  excluding  swallowed 
sputum,  the  presence  of  the  tubercle  bacillus  is  less 
significant  in  the  feces  than  in  other  material.  It  may, 
however,  be  taken  as  evidence  of  intestinal  tuberculosis 
when  clinical  signs  indicate  an  intestinal  lesion  and 
reasonable  care  is  exercised  in  regard  to  the  sputum. 
Success  in  the  search  will  depend  largely  upon  careful 
selection  of  the  portion  examined.  A  random  search 
will  almost  surely  fail.  Whitish  or  grayish  flakes  of 
mucus  or  blood-stained  or  purulent  particles  should  be 
spread  upon  slides  or  covers  and  stained  by  the  method 
given  upon  p.  236.  In  the  case  of  rectal  ulcers,  swabs 
can  be  made  directly  from  the  ulcerated  surface. 
With  young  children,  who  swallow  all  their  sputum, 
an  examination  of  the  stool  for  tubercle  bacilli  may 
be  the  means  of  diagnosing  tuberculosis  of  the  lung. 

6.  Crystals. — Various  crystals  may  be  found,  but 
few  have  any  significance.  Slender,  needle-like  crystals 
of  fatty  acids  and  soaps  (see  Fig.  49)  and  triple  phos- 
phate  crystals    (see    Fig.    152)    are    common.     Char- 


MICROSCOPIC    EXAMINATION  443 

acteristic  octahedral  crystals  of  calcium  oxalate  (see 
Fig.  51)  appear  after  ingestion  of  certain  vegetables. 
Charcot-Leyden  crystals  (see  Fig.  i8)  are  not  infre- 
quently encountered,  and  strongly  suggest  the  presence 
of  intestinal  parasites.  Yellowish  or  brown,  needle- 
like or  rhombic  crystals  of  hematoidin  (see  Fig.  49) 
may  be  seen  after  hemorrhage  into  the  bowel.  The 
dark  color  of  the  feces  after  administration  of  bismuth 
salts  is  due  largely  to  great  numbers  of  bismuth  sub- 
oxid  crystals.     They  resemble  hemin  crystals. 

7.  Parasites  and  Ova. — Descriptions  will  be  found 
in  the  following  chapter.  The  ova  most  likely  to  be 
encountered  are  shown  in  Figs.  178  and  182.  The 
flagellates  are  usually  best  found  in  the  second  stool 
after  a  saline  cathartic,  the  first  stool  being  ordinarily 
too  solid  and  the  later  ones  too  dilute. 

To  find  ova  when  scarce,  they  must  be  concentrated. 
Stiles  advises  thoroughly  mixing  the  stool  with  a  quart 
or  more  of  water,  allowing  to  settle,  pouring  ofT  the 
water  almost  down  to  the  sediment,  and  repeating  the 
process  as  long  as  any  matter  floats.  The  final  sedi- 
ment is  poured  into  a  conical  glass  and  allowed  to 
settle.  Ova  will  be  found  in  the  fine  sediment,  which 
can  readily  be  removed  with  a  pipet.  The  same  end 
may  be  accomplished  more  efficiently  and  more  quickly 
by  means  of  the  centrifuge;  but  one  must  learn  how  long 
his  individual  centrifuge  requires  to  throw  down  the 
ova  while  the  lighter  particles  still  float.  These  methods 
are  more  satisfactory  if  the  larger  particles  are  first  re- 
moved by  passing  the  fecal  suspension  through  two  or 
three  layers  of  gauze  or  through  a  succession  of  wire 
screens  with  mesh  apertures  ranging  from  6  to  100  to 


444  THE   FECES 

the  inch.  Such  concentration  methods  are  greatly  to 
be  preferred  to  direct  microscopic  examination  of  the 
stool;  not  only  are  the  ova  concentrated,  but  they  are 
more  easily  identified  than  in  untreated  feces,  since 
bacteria  and  debris  which  would  otherwise  obscure 
them  have  been  removed.  Other  and  more  complicated 
methods  have  been  devised,  but  those  just  given 
and  Pepper's  method  for  hookworm  eggs  (see  p.  505) 
will  probably  answer  all  clinical  needs. 

IV.  FUNCTIONAL  TESTS 

1.  Schmidt's  Test  Diet. — Much  can  be  learned  of 
the  various  digestive  functions  from  a  microscopic 
study  of  the  feces,  especially  when  the  patient  is  upon  a 
known,  diet.  For  this  purpose  the  standard  diet  of 
Schmidt  is  generally  adopted.     This  consists  of: 

Morning 0.5  liter  milk  and  50  Gm.  toast. 

Forenoon 0.5  liter  porridge,  made  as  follows:  40  Gm. 

oatmeal,  10  Gm.  butter,  200  c.c.  milk, 
300  c.c.  water,  one  egg,  and  salt  to  taste. 

Midday 125  Gm.  hashed  meat,  with  20  Gm.  butter, 

fried  so  that  the  interior  is  quite  rare; 
250  Gm.  potato,  made  by  cooking  190 
Gm.  potato  with  100  c.c.  milk  and  10  Gm. 
butter,  the  whole  boiled  down  to  250  c.c. 

Afternoon Same  as  morning. 

Evening Same  as  forenoon. 

At  the  beginning  of  the  diet,  the  stool  should  be 
marked  off  with  carmin  or  charcoal  (see  p.  447).  One 
should  familiarize  himself  with  the  feces  of  normal 
persons  upon  this  diet.  A  portion  of  the  stool  about 
the  size  of  a  walnut  should  be  rubbed  up  with  water 
to  a  consistency  of  thick  soup  and  examined  macro- 


FUNCTIONAL   TESTS  445 

scopically  and  microscopically.  The  microscopic  ex- 
amination may  be  facilitated  by  preparing  four 
slides:  one  of  the  diluted  feces  untreated;  one  treated 
with  dilute  Lugol's  solution;  one  with  30  per  cent, 
acetic  acid;  one  with  Sudan  III. 

Deficiency  of  starch  digestion  is  recognized  by  the 
number  of  starch-granules  which  strike  a  blue  color  with 
iodin.  With  exception  of  those  inclosed  in  plant  cells 
none  are  present  normally. 

The  degree  of  protein  digestion  is  ascertained  by  the 
appearance  of  the  muscle-fibers.  Striations  are  clearly 
visible  on  any  considerable  number  of  the  fibers  only 
when  digestion  is  imperfect  (see  Fig.  154).  They  are 
most  clearly  seen  in  the  acetic-acid  preparation.  The 
striations  usually  disappear  after  the  feces  have  stood 
for  some  time.  According  to  Schmidt,  the  presence  of 
nuclei  in  muscle-fibers  denotes  complete  absence  of 
pancreatic  function.  The  presence  of  connective- 
tissue  shreds  indicates  deficient  gastric  digestion, 
since  raw  connective  tissue  is  digested  only  in  the 
stomach.  These  shreds  can  be  recognized  macro- 
scopically  by  examining  in  a  thin  layer  against  a  black 
background,  and  microscopically  by  their  fibrous 
structure  and  the  fact  that  they  swell  up  and  become 
clear  and  gelatinous  when  treated  with  acetic  acid. 
The  only  structure  likely  to  cause  confusion  is  elastic 
tissue  and  this  is  rendered  more  distinct  by  acetic 
acid. 

Digestion  of  fats  is  checked  up  by  the  amount  of 
neutral  fat,  which  should  not  be  present  in  appreciable 
quantity  normally.  It  is  best  seen  after  staining  with 
Sudan   III.     The   amount  of  fatty   acid   seen  in  an 


446  THE    FECES 

acetic  acid  preparation  after  it  has  been  heated  until 
bubbles  rise  is  also  a  good  guide  if  one  is  familiar  with 
what  to  expect  under  normal  conditions. 

Schmidt's  nuclei  test  for  pancreatic  insufficiency  con- 
sists in  the  administration  of  a  ^^-cm.  cube  of  beef  or, 
better,  of  thymus  tied  in  a  little  gauze  bag  with  the 
test-meal.  The  meat  must  previously  have  been  hard- 
ened in  alcohol  and  well  washed  in  water.  When  the 
bag  appears  in  the  feces  it  is  opened  and  its  contents 
examined  microscopically  by  pressing  out  small  bits 
between  a  slide  and  cover.  A  drop  of  some  nuclear 
stain  may  be  applied  if  desired.  If  the  nuclei  are  for 
the  most  part  undigested,  pancreatic  insufficiency 
may  be  assumed,  since  it  is  probable  that  nuclei  can 
be  digested  only  by  the  pancreatic  juice.  Normally 
the  nuclei  are  digested,  provided  the  time  of  passage 
through  the  intestine  is  not  less  than  six  hours.  Upon 
the  other  hand,  if  the  time  of  passage  exceeds  thirty 
hours  nuclei  may  be  partially  digested  in  the  com- 
plete absence  of  pancreatic  juice. 

2.  Sahli's  Glutoid  Test. — The  Schmidt  test  diet  in- 
volves some  inconvenience  for  the  patient,  and  inter- 
pretation of  results  requires  much  experience  upon  the 
part  of  the  physician.  A  number  of  other  methods  of 
testing  the  digestive  functions  have  been  proposed. 
The  glutoid  test  of  Sahli  is  one,  of  the  most  satisfactory. 
This  is  similar  to  his  desmoid  test  of  gastric  digestion 
described  on  page  421.  A  glutoid  capsule  containing 
0.15  Gm.  iodoform  is  taken  with  an  Ewald  breakfast. 
The  capsule  is  not  digested  by  the  stomach  fluid,  but 
is  readily  digested  by  pancreatic  juice.  Appearance  of 
iodin  in  the  saliva  and  urine  within  four  to  six  hours 


FUNCTIONAL    TESTS  447 

indicates  normal  gastric  motility,  normal  intestinal  di- 
gestion, and  normal  absorption.  Instead  of  iodoform, 
0.5  Gm.  salol  may  be  used,  salicyluric  acid  appearing 
in  the  urine  in  about  the  same  time.  For  tests  for  iodin 
and  salicyluric  acid,  see  pages*  421  and  422. 

Glutoid  capsules  are  prepared  by  soaking  gelatin 
capsules  in  10  per  cent,  formalin.  Sahli  states  that 
filled  capsules  can  be  purchased  of  A.  G.  Haussmann, 
in  St.  Gall,  Switzerland.  To  make  sure  that  the  cap- 
sules are  soluble  one  should  try  a  test  upon  oneself. 

3.  Motility. — Ordinarily,  with  adults  who  are  upon 
a  mixed  diet,  fifteen  to  thirty  hours  are  required  for  the 
passage  of  ingested  material  through  the  gastro- 
intestinal tract.  With  infants  the  time  is  about  one- 
third  as  long.  In  diarrheal  conditions  it  is  usually 
much  shortened,  unless  the  pathologic  process  is  in 
the  colon.  In  intestinal  stasis  it  may  be  much  pro- 
longed. The  time  of  passage  is  ascertained  by  giving 
0.5  Gm.  of  powdered  charcoal  or  0.3  Gm.  of  carmine 
in  a  capsule  with  a  meal  and  watching  for  the  re- 
sulting discolored  feces. 


CHAPTER  VI 
ANIMAL  PARASITES 

Animal  parasites  are  common  in  all  countries,  but  are 
especially  abundant  in  the  tropics,  where,  in  some  locali- 
ties, almost  every  native  is  host  for  one  or  more  species. 
Because  of  our  growing  intercourse  with  these  regions 
the  subject  is  assuming  increasing  importance  in  this 
country.  Many  parasites,  hitherto  comparatively  un- 
known here,  will  probably  become  fairly  common. 

Some  parasites  produce  no  symptoms,  even  when 
present  in  large  numbers.  Others  cause  very  serious 
symptoms.  It  is,  however,  impossible  to  make  a  sharp 
distinction  between  pathogenic  and  non-pathogenic 
varieties.  Parasites  which  cause  no  apparent  ill  effects 
in  one  individual  may,  under  certain  conditions,  produce 
marked  disturbances  in  another.  The  disturbances  are 
so  varied,  and  frequently  so  indefinite,  that  diagnosis 
can  rarely  be  made  from  the  clinical  symptoms.  It 
must  rest  upon  detection,  by  the  naked  eye  or  the  micro- 
scope, of  (a)  the  parasites  themselves,  (b)  their  ova  or 
larvae,  or  (c)  some  of  their  products. 

Unlike  bacteria,  the  great  majority  of  animal  para- 
sites multiply  by  means  of  alternating  and  differently 
formed  generations,  which  require  widely  dift'erent  con- 
ditions for  their  development.  The  few  exceptions  are 
chiefly  among  the  protozoa.  Multiplication  of  para- 
sites within  the  same  host  is  thus  prevented.     In  the 

448 


ANIMAL  PARASITES  449 

case  of  the  hookworm,  for  example,  there  is  no  increase 
in  the  number  of  worms  in  the  host's  intestine,  except 
through  reinfection  from  the  outside.  The  ova  are 
carried  out  of  the  intestine  and  the  young  must  pass  a 
certain  period  of  development  in  warm,  moist  earth 
before  they  can  again  enter  the  human  body  and  grow 
to  maturity.  This  also  explains  the  geographic  dis- 
tribution of  parasites.  The  hookworm  cannot  flourish 
in  cold  countries;  malaria  can  prevail  only  in  localities 
in  which  the  mosquito.  Anopheles,  exists,  and  then  only 
after  the  mosquitoes  have  become  infected  from  a  hu- 
man being. 

In  general,  this  alternation  of  periods  of  development 
takes  place  in  one  of  three  ways: 

1.  The  young  remain  within  the  original  host,  but 
travel  to  other  organs,  where  they  do  not  reach  matu- 
rity, but  lie  quiescent  until  taken  in  by  a  new  host.  A 
good  example  is  Trichinella  spiralis. 

2.  The  young  or  the  ova  which  subsequently  hatch 
pass  out  of  the  host,  and  either  (a)  go  through  a  simple 
process  of  growth  and  development  before  entering 
another  host,  as  is  the  case  with  the  hookworm,  or  {h) 
pass  through  one  or  more  free-living  generations,  the 
progeny  of  which  infect  new  hosts,  as  is  the  case  with 
Strongyloides  intestinalis. 

3.  The  young  or  ova  or  certain  specialized  forms 
either  directly  {e.g.,  malarial  parasites)  or  indirectly 
{e.g.,  tapeworms)  reach  a  second  host  of  different 
species,  where  a  widely  different  process  of  develop- 
ment occurs.  The  host  in  which  the  adult  or  sexual 
existence  is  passed  is  called  the  definitive  or  final  host; 
that  in  which  the  intermediate  or  larval  stage  occurs,  the 


450  ANIMAL    PARASITES 

intermediate  host.  Man,  for  example,  is  the  definitive 
host  for  TcBnia  saginata,  and  the  intermediate  host  for 
the  malarial  parasites  and  Tcenia  echinococcus . 

At  this  place  a  few  words  concerning  the  classification 
and  nomenclature  of  living  organisms  in  general  will 
be  helpful.  Individuals  which  are  alike  in  all  essential 
respects  are  classed  together  as  a  species.  Closely  re- 
lated species  are  grouped  together  to  form  a  genus; 
genera  that  have  certain  characteristics  in  common 
make  up  a  family;  families  are  grouped  into  orders;  or- 
ders, into  classes;  and  classes,  finally,  into  the  branches 
or  phyla,  which  make  up  the  animal  and  vegetable  king- 
doms. In  some  cases  these  groups  are  subdivided  into 
intermediate  groups — subphyla,  subfamilies,  etc.,  and 
occasionally  slight  differences  warrant  subdivision  of 
the  species  into  varieties. 

The  scientific  name  of  an  animal  or  plant  consists  of 
two  parts,  both  Latin  or  Latinized  words,  and  is  printed 
in  italics.  The  first  part  is  the  name  of  the  genus  and 
begins  with  a  capital  letter;  the  second  is  the  name  of  the 
species  and  begins  with  a  lower  case  letter,  even  when  it 
was  originally  a  proper  name.  When  there  are  varieties 
of  a  species,  a  third  part,  the  designation  of  the  variety, 
is  appended.  The  author  of  the  name  is  sometimes  in- 
dicated in  Roman  type  immediately  after  the  name  of 
the  species.  Examples:  Spirochceta  vincenti,  often  ab- 
breviated to  Sp.  vincenti  when  the  genus  name  has  been 
used  just  previously;  Staphylococcus  pyogenes  alhus; 
Necator  americanus,  Stiles. 

At  the  present  time  there  is  great  confusion  in  the 
naming  and  classification  of  parasites.  Some  have  been 
given  a  very  large  number  of  names  by  different  ob- 


PHYLUM   PROTOZOA  45 T 

servers,  and  in  many  cases  different  parasites  have  been 
described  under  the  same  name.  The  alternation  of 
generations  and  the  marked  differences  in  some  cases 
between  male  and  female  have  contributed  to  the  con- 
fusion, different  forms  of  the  same  parasite  being  de- 
scribed as  totally  unrelated  species. 
-  The  number  of  parasites  which  have  been  described 
as  occurring  in  man  and  the  animals  is  extremely  large. 
Only  those  which  are  of  medical  interest  are  mentioned 
here.  They  belong  to  four  phyla — Protozoa,  Platyhel- 
minthes,  Nemathelminthes,  and  Arthropoda. 

PHYLUM  PROTOZOA 

These  are  unicellular  organisms,  the  simplest  types 
of  animal  life.  There  is  very  little  differentiation  of 
structure.  Each  contains  at  least  one,  and  some  sev- 
eral, nuclei.  Some  contain  contractile  vacuoles;  some 
have  cilia  or  flagella  as  special  organs  of  locomotion. 
They  reproduce  by  division,  by  budding,  or  by  sporula- 
tion.  Sometimes  there  is  an  alternation  of  generations, 
in  one  of  which  sexual  processes  appear,  as  is  the  case 
with  the  malarial  parasites.  The  protozoa  are  very 
numerous,  the  subphylum  Sarcodina  alone  including 
no  less  than  5000  species.  Most  of  the  protozoa  are 
microscopic  in  size;  a  few  are  barely  visible  to  the  naked 
eye.  The  beginning  student  can  gain  a  general  idea  of 
their  appearance  by  examining  water  (together  with  a 
little  of  the  sediment)  from  the  bottom  of  any  pond. 
Such  water  usually  contains  amebai  and  a  considerable 
variety  of  ciliated  and  flagellated  forms. 

The  following  is  an  outline  of  those  protozoa  which 


452 


ANIMAL  PARASITES 


are  of  medical  interest,  together  with  the  subphyla  and 
classes  to  which  they  belong: 

PHYLUM  PROTOZOA 

SuBPHYLUM  I.  S  ARC  ODIN  A. — Locomotion  by  means  of 
pseudopodia. 

Class  Rhizopoda. — Pseudopodia  form  lobose  or  reticulose  processes. 

Genus  Species 

Endamceba.  E.  histolytica. 

E.  coli. 
E.  gingivalis. 

SuBPHYLUM  XL  MASTIGOPHORA  (FLA GELLATA).— Locomotion 
by  means  of  flagella. 

Class  Zoomastigophora. — Forms  in  which  animal  characteristics 
predominate. 


Genus 

Species 

Spirochaeta. 

Sp.  recti  rrentis. 

Sp.  vincenti. 

Sp.  buccalis. 

Sp.  dentium. 

Sp.  refringens. 

Treponema.^ 

T.  pallidum. 

T.  pertenue. 

Trypanosoma. 

T.  gambiense. 

T.  rhodesiense. 

T.  cruzi. 

T.  lewisi. 

T.  evansi. 

T.  brucei. 

T.  equiperdum, 

Leishmania. 

L.  donovani. 

L.  tropica. 

L.  infantum. 

Cercomonas. 

C.  ho  minis 

Bodo. 

B.  urinarius. 

Trichomonas 

T.  intestinalis. 

T.  vaginalis. 

T.  pulmonalis. 

Lamblia. 

L.  intestinalis. 

PHYLUM    PROTOZOA  453 

SuBPHYLUM  III.     SPOROZOA. — All  members  parasitic.   Propaga- 
tion by  means  of  spores.     No  special  organs  of  locomotion. 
Class  Telosporidia. — Sporulation  ends  the  life  of  the  individual. 

Genus  Species 

Coccidium.  C.  cuniculi. 

Plasmodium.  P.  vivax. 

P.  malariae. 

P.  falciparum. 
Babesia.  B.  bigeminum. 

SuBPHYLUM  IV.    INFUSORIA. — Locomotion  by  means  of  cilia. 
Class  Ciliata. — Cilia  present  throughout  life. 

Genus  Species     - 

Balantidium.  B.  coli. 

B.  minutum 

SUBPHYLUM  SARCODINA 
Class  Rhizopoda 

These  are  protozoa  the  body  substance  of  which 
forms  changeable  protoplasmic  processes,  or  pseudo- 
podia,  for  the  taking  in  of  food  and  for  locomotion. 
They  possess  one  or  several  nuclei. 

1.  Genus  Endamoeba. — i.  Endamceba  histolytica. 
— This  organism  is  found,  often  in  large  numbers,  in 
the  stools  of  tropical  dysentery  and  in  the  pus  and  walls 
of  hepatic  abscesses  associated  with  dysentery.  Infec- 
tion is  more  common  in  this  country  than  was  at  one 
time  supposed  and  has  even  been  reported  in  the 
Northern  States.  The  parasite  is  a  grayish  or  color- 
less, granular  cell,  usually  between  25  and  40  /x  in 
diameter  (Fig.  155).  Its  appearance  varies  accord- 
ing to  its  stage  of  development.  In  the  vegetative 
stage,  which  is  found  in  acute  dysentery,  there  is  a 
distinct,  homogeneous,  refractile  ectoplasm  and  a  granu- 


454 


ANIMAL    PARASITES 


lar  endoplasm  containing  one  or  more  distinct  vacuoles, 
a  lound  nucleus  which  is  ordinarily  very  indistinct,  and, 
frequently,  ingested  red  blood-corpuscles  and  bacteria. 
When  at  rest  its  shape  is  spheric,  but  upon  a  warm  slide 
it  exhibits  the  characteristic  ameboid  motion,  constantly 
changing  its  shape  or  moving  actively  about  by  means 
of  distinct  pseudopodia.  This  motion  is  its  most  dis- 
tinctive feature,  and  should  always  be  seen  to  establish 


Fig.   155. — Endamasba    hislul\iu,i    \\\    iniL'suiial    mucui 
corpuscles  and  bacteria  (Losch). 


with    blood- 


the  identity  of  the  organism  in  this  stage.  It  is  lost 
when  the  specimen  cools,  and  can  usually  not  be  re- 
established by  warming.  If  neutral  red  in  0.5  per  cent, 
solution  be  run  under  the  cover-glass,  it  will  be  taken 
up  by  the  endamebae  and  other  protozoa  and  will  render 
them  conspicuous  without  killing  them  ("vital 
staining"). 

In  dysentery  "carriers"  and  in  chronic  cases  when 
the  stools  are  formed  and  hard,  most  or  all  of  the  para- 


PHYLUM    PROTOZOA  455 

sites  may  become  encysted.  Their  appearance  in  this 
stage  of  development  is  given  in  the  table  on  pages 
456,  457.  The  structure  of  the  cysts  is  best  seen  when 
a  drop  of  Lugol's  solution  is  mixed  with  the  fecal 
matter  on  the  slide. 

When  the  presence  of  endamebaj  is  suspected,  the 
stool  should  be  passed  into  a  warm  vessel  and  kept 
warm  until  and  during  the  examination.  A  warm  stage 
can  be  improvised  from  a  plate  of  copper  with  a  hole 
cut  in  the  center.  This  is  placed  upon  the  stage  of  the 
microscope,  and  one  of  the  projecting  ends  is  heated 
with  a  small  flame.  Endamebae  are  most  likely  to  be 
found  in  grayish  or  blood-streaked  particles  of  mucus. 
Craig  recommends  the  liquid  stool  following  a  saline 
cathartic.  Favorable  material  for  xamination  can 
often  be  obtained  at  one's  convenience  by  inserting  into 
the  rectum  a  large  catheter 'with  roughly  cut  lateral 
openings.  A  sufficient  amount  of  mucus  or  fecal  matter 
will  usually  be  brought  away  by  it. 

No  staining  method  is  as  useful  in  diagnosis  as  the 
study  of  the  living  and  moving  parasite.  For  more 
detailed  study,  Darling  recommends  the  following 
method:  Stain  with  Wright's  (or  Hastings'  or  Leish- 
man's)  stain  in  the  usual  way,  and  follow  this  with 
Giemsa's  stain,  diluted  i  :  lo,  until  the  film  has  a  purple 
cast.  Then  plunge  the  preparation  into  a  small  beaker 
of  60  per  cent,  alcohol  to  which  lo  to  20  drops  of 
ammonia  have  been  added  and  keep  it  in  motion  until 
the  desired  differentiation  is  obtained,  when  the  film 
will  have  a  violet  color. 

2.  Endamceba  coli. — This  organism,  which  is  fre- 
quently found  in  the  stools  of  healthy  persons,  is  simi- 


456  ANIMAL   P ABASHES 

lar  to  E.  histolytica,  but  is  smaller,  rarely  over  25  /i 
in  diameter.  It  has  less  distinct  pseudopodia,  less 
sharp  differentiation  between  ectoplasm  and  endoplasm, 
less  active  motion,  and  more  distinct  nucleus,  and  does 
not  contain  ingested  red  corpuscles  or  never  more  than 
one  or  two.  The  vacuoles  contain  glycogen-granules 
which  stain  brown  with  Lugol's  solution;  such  granules 
are  rare  in  E.  histolytica. 

The  principal  points  of  distinction  between  E.  histo- 
lytica and  E.  coli  are  included  in  the  following  table 
which  is  slightly  modified  from  Craig^ : 

VEGETATIVE  STAGE 

This  stage  of  E.  histolytica  is  found  in  acute  dysentry. 

Endamceba  histolytica  Endamceba  coli 

Averages  larger.     Unimportant.        Averages  smaller. 

Actively  motile.       Characteris-        Sluggishly      motile.       Seldom 
tic.     Often  moves  from  place  to     moves  from  place  to  place, 
place. 

Ectoplasm    hyaline,    glass-like,        Ectoplasm  not  glass-like,  poorly 
sharply  differentiated  from  endo-    differentiated  from  endoplasm. 
plasm.     Characteristic. 

Nucleus  usually  indistinct,  often        Nucleus  distinct.  Located  near 
invisible.     Changes  position  with     center, 
motion  of  parasite. 

Red  blood-cells  present  in  endo-  No  red  blood-cells  (or  never 
plasm  when  stool  contains  blood,  more  than  one  or  two)  in  endo- 
Very  characteristic.  plasm  when  stool  contains  blood. 

PRECYSTIC  STAGE 

E.  histolytica  may  be  found  in  this  stage  when  symp- 
toms of  dysentery  have  practically  disappeared.     The 
*  Craig:  Archives  of  Internal  Medicine,  1914,  xiii,  917. 


PHYLUM   PROTOZOA  457 

parasite  is  reduced  in  size,  is  sluggishly  motile,  and 
becomes  practically  indistinguishable  from  E.  coli.  The 
distinction  must  be  based  upon  the  vegetative  or  cystic 
forms,  a  few  of  which  can  usually  be  found  in  the  same 
stool. 

CYSTIC  STAGE 

In  formed  stools  both  endamebae  are  commonly  en- 
cysted. This,  therefore,  is  the  form  of  E.  histolytica 
to  be  looked  for  between  recurrences  and  in  dysentery 
"carriers." 

Endamceha  histolytica  Endammba  coli 

Cysts    spheric    or    oval.     Cyst        Similar,  but  double  outline  of 

wall  single  and  delicate  in  young  wall    more    frequently   observed 

cysts;     thicker     and     sometimes  and  more  distinct, 
double  outlined  in  older  ones. 

Diameter  10-20  fi;  average,  12  /z.        Diameter  10-25  m;  average,  15  ft. 

Cytoplasm  of  young  cysts  granu-         Similar,    but   chromidia    very 
lar,    often    with    a  large  vacuole,    rare. 
Presence  of    chromidia    (brightly 
refractive,    spindle-shaped    or    ir- 
regular masses  of  chromatin)  char- 
acteristic. 

Fully   developed   cysts   contain        Fully  developed  cyst  contains 

four  distinct  nuclei  seen  by  focus-  eight  to  sixteen  nuclei,  eight  being 

ing  at  different  levels.     Very  char-  the  normal  number, 
acteristic. 

3.  Endamoeba  gingivalis. — That  endamebas  are 
common  in  the  mouth  and  about  the  teeth  has  long 
been  recognized,  but  they  have  generally  been  regarded 
as  harmless  or  even  as  beneficial  because  they  feed  ex- 
tensively upon  bacteria.  There  is  apparently  only  one 
species,  which  has  been  variously  called  E.  buccalis,  E. 
dentalis,  and  E.  gingivalis,  the  last  name  being  now 


458 


ANIMAL   PARASITES 


accepted  as  correct.  Within  the  past  few  years  it  has 
attracted  much  attention  as  the  possible  cause  of  pyor- 
rhea alveolaris.  The  organisms  are  found  in  the  le- 
sions of  practically  every  case  of  pyorrhea,  often  in 


Fig.  156. — Endamaeba  gingivalis,  pus-corpuscles,  red  blood-cells, 
spirochetes,  and  bacteria  in  a  smear  from  a  lesion  of  pyorrhea  alveo- 
laris. Giemsa's  slain,  without  alkali,  twelve  hours  (  X  850).  The 
figure  shows  three  endamebae,  each  with  one  round  nucleus  (red). 
The  cytoplasm  (deep  sky  blue)  contains  vacuoles  and  bacteria.  The 
largest  parasite  contains  ten  nuclei  (blackish-purple)  from  ingested 
cells.  A  digestion  vacuole  is  seen  at  each  end  of  the  long  bacillus  in 
the  endameba  near  the  bottom.  The  red  corpuscles  were  salmon 
colored;  nuclei  of  leukocytes,  reddish-purple;  spirochetes,  bluish- 
purple. 


large  numbers.  In  some  parts  of  the  slide  from  which 
Fig.  156  was  made,  there  were  as  many  as  20  in  a 
single  field  of  the  oil-immersion  lens.  Upon  the  other 
hand,  a  few  are  often  found  between  the  gums  and 
teeth   when    no    lesions    are    recognizable.     The    evi- 


PHYLUM    PROTOZOA  45Q 

dence  at  present  available  suggests  that  the  organism 
is  a  factor  in  the  etiology  of  pyorrhea,,  but  the  claim 
that  it  is  the  sole  specific  cause  is  not  warranted. 

Material  is  obtained  for  study  by  scraping  between 
the  teeth  and  the  gum  with  a  sterile  wooden  toothpick. 
When  pus-pockets  exist,  the  bottom  and  side  of  a  pocket 
should  be  scraped  with  a  dental  scaler.  This  material 
may  be  examined  in  the  fresh  state  by  mixing  it  with 
a  little  saliva  and  placing  on  a  warmed  slide.  The  or- 
ganism is  less  active  than  E.  histolytica,  more  so  than 
E.  coli.  Unless  motion  is  seen  it  will  be  difficult  to 
recognize.     Individuals  range  in  size  from  lo  to  35  fj.. 

In  general,  the  endamebae  are  more  easily  identified 
in  stained  smears.  The  smears  are  made  by  streaking 
the  toothpick  three  or  four  times  across  the  slide. 
Often  one  of  the  streaks  will  contain  many  of  the 
parasites  and  the  others  only  a  few.  Giemsa's  solu- 
tion, applied  as  described  for  blood  (see  p.  313)  but 
allowed  to  act  for  three  to  twelve  hours,  is  the  most 
satisfactory  stain.  With  this,  the  cytoplasm  of  end- 
amebae is  blue  and  shows  the  vacuoles  clearly,  the  small 
round  nucleus  is  red,  ingested  bacteria  purple  and  nuclei 
of  ingested  cells  deep  purple.  In  such  preparations  it 
is  well-nigh  impossible  to  mistake  pus  and  epithelial 
cells  for  endamebae.  Wright's  stain  gives  a  similar 
picture  but  the  differentiation  is  somewhat  less  sharp. 
The  writer  has  found  pyronin  methyl  green  (see  p.  642) 
to  be  fairly  satisfactory.  It  stains  the  cytoplasm  of 
endamebae  red. 

4.  Other  Endamebae. — E.  tetragena,  which  was  de- 
scribed in  1907  by  Viereck,  is  now  regarded  as  identical 
with  E.  histolytica.     A  number  of  similar  organisms  have 


460  ANIMAL    PARASITES 

been  described  as  occurring  in  pus  and  in  ascitic  and 
other  body  fliyds,  but  it  is  probable  that  in  many  cases, 
at  least,  the  structures  seen  were  ameboid  body  cells. 

SUBPHYLUM  MASTIGOPHORA  (FLAGELLATA) 
Class  Zoomastigophora 

The  protozoa  of  this  subphylum  are  provided  with  one 
or  several  whip-like  appendages  with  lashing  motion, 
termed  flagella,  which  serve  for  locomotion  and,  in 
some  cases,  for  feeding.  They  generally  arise  from  the 
anterior  part  of  the  organism.  Some  members  of  the 
group  also  possess  an  undulating  membrane — a  delicate 
membranous  fold  which  extends  the  length  of  the  body 
and  somewhat  suggests  a  fin.  When  in  active  motion 
this  gives  the  impression  of  a  row  of  cilia.  The  fiagel- 
lata  do  not  exhibit  ameboid  motion,  and,  in  general, 
maintain  an  unchanging  oval  or  spindle  shape,  and  con- 
tain a  single  nucleus.  The  cytoplasm  contains  nu- 
merous granules  and  usually  several  vacuoles,  one  or 
more  of  which  may  be  contractile.  Encystment  as  a 
means  of  resisting  unfavorable  conditions  is  common. 

1.  Genus  Spirochaeta. — The  spirochetes  appear  to 
occupy  a  position  midway  between  the  bacteria  and 
protozoa,  but  are  more  frequently  described  with  the 
latter.  They  are  receiving  much  atten  tion  at  the  present 
time  and  the  appreciation  of  their  importance  is  grow- 
ing rapidly. 

I.  Spirochaeta  recurrentis. — This  spirochete  was 
described  by  Obermeier  as  the  cause  of  relapsing  fever. 
It  appears  in  the  circulating  blood  during  the  febrile 
attack,  and,  unlike  the  malarial  parasite,  lives  in  the 
plasma   without   attacking   the   red   corpuscles.     The 


PHYLUM   PROTOZOA 


461 


organism  is  an  actively  motile  spiral,  15  to  20  /x  long, 
with  three  to  twelve  wide,  fairly  regular  turns.  It  can 
be  seen  in  fresh  unstained  blood  with  a  high  dry  lens, 
being  located  by  the  commotion  which  it  creates  among 
the  red  cells.  For  diagnosis,  thin  films,  stained  with 
Wright's  or  some  similar  blood-stain,  are  used  (Fig. 
157).  In  such  preparations  the  spirals  are  not  so 
regular. 


Fig.  157- 


-Spirochete  of  relapsing  fever  in  blood  (  X  looo)  (Karg  and 
Schmorl). 


It  is  generally  believed  that  relapsing  fever  does  not 
occur  in  the  United  States,  but  Meader  has  recently 
reported  five  cases  which  originated  in  Colorado. 
Spirochetes  from  one  of  these  cases  are  shown  in  Plate 
VI. 

Besides  Spirochceta  recurrentis,  a  number  of  distinct 
strains  have  been  described  in  connection  with  different 
types  of  relapsing  fever:  Sp.  novyi,  Sp.  kochi,  Sp. 
duttoni,  and  Sp.  carteri. 


462  ANIMAL   PARASITES 

2.  Spirochaeta  vincenti.- -In  stained  smears  from  the 
ulcers  of  Vincent's  angina  (see  p.  539)  are  found  what 
appear  to  be  two  organisms.  One,  the  "fusiform  bacil- 
lus," is  a  slender  rod,  4  to  8  /x  long,  pointed  at  both 
ends,  and  sometimes  curved.  The  other  is  a  slender 
spiral  organism,  10  to  20  n  long,  with  three  to  ten  com- 
paratively shallow  turns  (see  Fig.  216).  These  were 
formerly  thought  to  be  bacteria,  a  spirillum  and  a  bacillus 
living  in  symbiosis.  The  present  tendency  is  to  regard 
them  as  stages  or  forms  of  the  same  organism,  and  to 
class  them  among  the  spirochetes.  The  same  organ- 
isms are  quite  constantly  present  in  large  numbers 
in  ulcerative  stomatitis  and  in  noma.  They  are 
not  infrequently  found  in  small  numbers  in  normal 
mouths. 

3.  Other  Spirochetes. — A  number  of  harmless  forms 
are  of  interest  because  of  the  possibility  of  confusing 
them  with  the  more  important  pathogenic  varieties. 
Of  these,  Sp.  buccalis  and  Sp.  dentium  are  inhabitants. 
of  the  normal  mouth.  When  the  teeth  and  gums  are 
not  in  good  condition  they  are  often  found  in  immense 
numbers  (see  Fig.  156).  The  former  is  similar  in 
morphology  to  Sp.  vincenti.  Sp.  dentium  (Fig.  158) 
is  smaller  (4  to  10  /x),  more  delicate,  has  deep  curves, 
and  may  be  easily  mistaken  for  Treponema  pallidum. 
It,  also,  stains  reddish  with  Giemsa's  stain.  In  sus- 
pected syphilitic  sores  of  the  mouth  it  is,  therefore, 
important  to  make  smears  from  the  tissue  juices 
rather  than  from  the  surface  (see  p.  550).  Sp. 
refringens  is  frequently  present  upon  the  surface  of 
ulcers,  especially  about  the  genitals,  and  has  doubtless 
many  times  been  mistaken  for  Treponema  pallidum.     It 


PHYLUM    PROTOZOA  463 

can  be  avoided  by  properly  securing  the  material  for 
examination;  but  its  morphology  should  be  sufficient  to 
prevent  confusion.  It  is  thicker  than  the  organism  of 
syphilis,  stains  more  deeply,  and  has  fewer  and  shallower 
curves  (Figs.  158  and  222).  Giemsa's  stain  gives  it  a 
bluish  color. 

'  Castellani  has  called  attention  to  a  bronchial  spiro- 
chetosis and  his  observations  have  been  confirmed  by 
other  workers  in  Europe,  Asia,  and  the  Philippines. 
When  chronic  the  condition  resembles  tuberculosis  but 


Fig.  158. — Spiral  organisms:  A,  Treponema  pallidum:  B,  Spiro- 
chcBla  refringens;  C,  Spirochcela  denlinm.  Two  red  corpuscles  are  also 
shown  (  X  1200). 

is  distinguished  by  finding  spirochetes  in  the  sputum. 
Infectious  jaundice  is  also  now  known  to  be  caused  by 
a  spirochete,  Sp.  iclerohemorrhagicce.  This  parasite  has 
recently  been  found  in  the  blood  and  organs  oi  wild  rats 
in  various  parts  of  this  country  and  Europe. 

2.  Genus  Treponema. —  i.  Treponema  pallidum. 
— This  is  the  organism  of  syphilis.  Its  description 
and  methods  of  diagnosis  will  be  found  on  pp.  548-553. 

2.  Treponema  pertenue,  morphologically  very  simi- 
lar to  Treponema  pallidum,  was  found  by  Castellani  in 
yaws,  a  skin  disease  of  the  tropics. 


464  ANIMAL    PARASITES 

3.  Qenus  Trypanosoma. — Trypanosomes  have 
been  found  in  the  blood-plasma  of  a  great  variety  of 
vertebrates.  Many  of  them  appear  to  produce  no 
symptoms,  but  a  few  are  of  great  pathologic  importance. 
As  seen  in  the  blood,  they  are  elongated,  spindle-shaped 
bodies,  the  average  length  of  different  species  varying 
from  10  to  70  fi.  Along  one  side  there  runs  a  delicate 
undulating  membrane,  the  free  edge  of  which  appears 
to  be  somewhat  longer  than  the  attached  edge,  thus 
throwing  it  into  folds.  Somewhere  in  the  body, 
usually  near  the  middle,  is  a  comparatively  pale- 
staining  nucleus;  and  near  the  posterior  end  is  a  smaller, 
more  deeply  staining  chromatin  mass,  the  micronucleus 
or  blepharoplast.  A  number  of  coarse,  deeply  staining 
granules,  chromatophores,  may  be  scattered  through 
the  cytoplasm.  A  flagellum  arises  in  the  blepharoplast, 
passes  along  the  free  edge  of  the  undulating  membrane, 
and  is  continued  anteriorly  as  a  free  flagellum.  These 
details  of  structure  are  well  shown  in  Plate  VI. 

The  life  history  of  the  trypanosomes  is  not  well 
known.  In  most  cases  there  is  an  alternation  of  hosts, 
various  insects  playing  the  part  of  definitive  host. 

Trypanosomes  have  been  much  studied  of  late,  and 
many  species  have  been  described.  At  least  three  have 
been  found  in  man. 

Trypanosoma  gambiense  is  the  parasite  of  African 
"sleeping  sickness."  Its  detection  in  the  blood  is  de- 
scribed on  page  348.  It  is  more  abundant  in  the  juice 
obtained  by  aspirating  a  lymph  gland  with  a  large 
hypodermic  needle,  and  in  the  late  stages  is  also  found 
in  the  cerebrospinal  fluid.  A  new  species  causing 
sleeping  sickness  in  man  has  recently  been  described  and 


PHYLUM   PROTOZOA  465 

has  been  named  T.  rhodesiense.  The  chief  point  of 
distinction  from  T.  gambiense  is  the  situation  of  the 
nucleus  close  to  or  even  posterior  to  the  blepharoplast. 
It  is  transmitted  by  the  fly  Glossina  morsitans. 

Trypanosoma  cruzi  is  a  small  form  which  has  been 
found  in  the  blood  of  man  in  Brazil. 

Try panosoma  lewisi,  a  very  common  and  apparently 
harmless  parasite  of  gray  rats,  especially  sewer  rats,  is 


Fig.   159. —  Trypanosoma   lewisi  in  blood  of  rat.     The  red  corpuscles 
were  decolorized  with  acetic  acid  (X  1000). 


interesting  because  it  closely  resembles  the  pathogenic 
forms,  and  is  easily  obtained  for  study.  Its  posterior 
end  is  more  pointed  than  that  of  T.  gambiense  (Fig. 

159)- 

Trypanosoma  evansi,  T.  brucei,  and  T.  equiperdum 
produce  respectively  surra,  nagana,  and  dourine,  which 
are  common  and  important  diseases  of  horses,  mules, 
and  cattle  in  the  Philippines,  East  India,  and  Africa. 


466  ANIMAL   PARASITES 

4.  Genus  Leish mania. — The  several  species  which 
compose  this  genus  are  apparently  closely  related  to  the 
trypanosomes,  but  their  exact  classification  is  undeter- 
mined. They  have  been  grown  outside  the  body  and 
their  transformation  into  flagellated  trypanosome-like 
structures  has  been  demonstrated.  Calkins  places 
them  in  the  genus  Herpetomonas. 

I.  Leishmania  donovani  is  the  cause  of  kala-azar,  an 
important  and  common  disease  of  India.  With 
Wright's  stain  the  "Leishman-Donovan  bodies"  are 
round  or  oval,  light  blue  structures,  2  to  3  /x  in  diameter, 
with  two  distinct  reddish  purple  chromatin  masses, 
one  large  and  pale  (trophonucleus) ,  the  other  small 


Pig.  160. — Cercomonas  hominis  (about  x  500):  A,  Larger  variety;  B, 
smaller  variety  (Davaine). 

and  deeply  staining  (blepharoplast) .  The  parasites 
are  especially  abundant  in  the  spleen,  splenic  puncture 
being  resorted  to  for  diagnosis.  They  are  readily 
found  in  smears  stained  by  any  of  the  Romanowsky 
methods.  Then  he  chiefly  within  endothelial  cells 
and  leukocytes.  They  are  also  present  within  leukocytes 
in  the  peripheral  blood,  but  are  difficult  to  find  in  blood- 
smears. 

2.  Leishmania  tropica  resembles  the  preceding.  It 
is  found,  lying  intracellularly,  in  the  granulation  tissue 
of  Delhi  boil  or  Oriental  sore 

3.  Leishmania  infantum  has  been  found  in  an  obscure 
form  of  infantile  splenomegaly  in  Algiers. 


PHYLUM   PROTOZOA  467 

5.  Genus   Cercomonas. — i.  Cercomonas  hominis, 

sometimes  found  in  the  feces,  particularly  in  tropical 
regions,  is  probably  harmless.  The  body  is  10  to  12  ju 
long,  is  pointed  posteriorly,  and  has  a  flagellum  at  the 
anterior  end  (Fig.  i6o).  The  nucleus  is  difficult  to 
make  out.  The  feces  should  be  examined  in  the  fresh 
state,  and  preferably  while  warm,  in  order  to  observe 
the  active  motion  of  the  organism. 

6.  Genus  Bodo. — i.  Bodo  urinarius  is  sometimes 
seen  in  the  urine,  darting  about  in  various  directions. 
It  is  probably  an  accidental  contamination.  It  has  a 
lancet-shaped  body,  about  10  n  long,  and  is  somewhat 
twisted  upon  itself,  with  two  flagella  at  the  end. 

7.  Genus  Trichomonas. — i.  Trichomonas  intes- 
tinalis  is  sometimes  confused  with  the  two  flagellates 
just  described  but  is  more  important  and  more  definitely 
known  than  either.  It  is  an  oval  or  pear-shaped  cell 
of  somewhat  changing  shape.  The  average  size  is 
about  10  by  15  M  although  there  is  considerable  variation 
among  individuals.  An  oval  nucleus  can  sometimes 
be  made  out  in  the  anterior  half  of  the  cell.  At  the 
anterior  or  blunt  end  there  is  a  cluster  of  three — some 
say  four — flagella  of  equal  length,  and  along  one  side  is 
an  undulating  membrane  the  thickened  free  edge  of 
which  is  continued  backward  as  a  short  flagellum. 
Owing  to  the  active  motion  of  the  flagella  and  undulat- 
ing membrane,  these  are  not  easily  seen,  and  at  first 
sight  the  parasite  has  much  the  appearance  of  a  pus- 
corpuscle  moving  busily  about  among  the  fecal  particles. 
According  to  Stitt  the  flagella  can  be  seen  more  clearly 
if  a  drop  of  Gram's  iodin  solution  be  added  to  the 
preparation  on  the  slide.     The  organism's  usual  habitat 


468  ANIMAL    PARASITES 

is  the  colon  where,  contrary  to  what  is  known  of  the 
similar  or  identical  T.  vaginalis,  it  is  said  to  prefer  an 
alkaline  medium.  Under  conditions  unfavorable  to 
active  life  it  becomes  encysted. 

Trichomonas  intestinalis  is  common  in  the  tropics,  and 
from  recent  reports  it  appears  to  be  widespread  through- 
out the  United  States.  While  it  was  formerly  believed 
to  be  non-pathogenic,  it  is  now  well-established  that 
it  may  cause  a  diarrhea  of  the  dysenteric  type  or  that  it 
at  least  may  greatly  aggravate  an  already  existing  in- 
flammatory condition.  The  para- 
sites are  often  so  abundant  that  four 
or  more  may  be  seen  in  a  single  field 
of  the  high  dry  objective. 

2.  Other  Trichomonads. — 
Various  forms  have  been  described, 
regarded  by  some  as  identical  with 

Pig.       161. — Tricho-    >r     '    .     .•      t      ■>  .t  j'i'i. 

monas  vaginalis  (about  T.  intesttnalts,  by  othcrs  as  distmct 
+  1000)  (after  Koiiiker  specics.     Among  these  are  T.  pul- 

and  Scanzoni).  .         °  ^ 

monalis,  which  has  been  encountered 
in  the  sputum  of  persons  sufi"ering  from  pulmonary  gan- 
grene and  putrid  bronchitis,  and  T.  vaginalis  which  is 
often  found  in  the  leucorrheal  discharge  of  catarrhal  vagi- 
nitis. The  latter  flourishes  only  in  an  acid  medium  and 
disappears  when  the  discharge  becomes  alkaline  as,  for 
instance,  during  menstruation.  In  a  case  of  coincident 
severe  inflammation  of  the  vagina  and  of  the  gums 
recently  studied  by  Lynch,  the  parasites  were  very 
numerous  in  both  situations.  They  averaged  22  by 
26  fi  in  size,  presented  four  flagella  at  the  anterior  end 
and  had  an  undulating  membrane  but  no  posterior 
flagellum.  Treatment  with  an  alkaline  wash  was  com- 
pletely successful. 


PHYLUM    PROTOZOA 


469 


A  few  cases  have  been  reported  in  which  T.  vaginalis 
was  apparently  the  cause  of  a  urethritis  in  the  male. 

8.  Genus  Lamblia. — i.  Lamblia  intestinalis  is  a 
very  common  parasite  in  the  tropics,  but  is  generally 
considered  of  little  pathogenic  importance.  It  ap- 
pears, however,  to  be  capable  of  producing  a  chronic 
diarrhea.     In  6000  stool   examinations  at   the  Mayo 


Fig.   162. — Lamblia  intestinalis  from  the  intestines  of  a  mouse  (about 
X  2000)    (Grassi  and  Schweiakoff). 


Clinic  this  parasite  was  found  66  times,  the  infected 
individuals  coming  chiefly  from  the  Northern  States 
and  Canada.  The  organism  is  pear-shaped,  measures 
10  to  15  /i  or  more  in  length,  and  has  a  depression  on  one 
side  of  the  blunt  end,  by  which  it  attaches  itself  to  the 
tops  of  the  epithelial  cells  of  the  intestinal  wall.  Three 
pairs  of  flagella  are  arranged  about  the  depression  and 
one  pair  at   the  pointed   end    (Fig.    162).     Its  usual 


470  ANIMAL   PARASITES 

habitat  is  the  upper  part  of  the  small  intestine.  Unless 
the  stool  is  obtained  by  catharsis  (see  p,  443),  encysted 
forms  only  may  be  found,  and  these  may  be  difficult 
or  impossible  to  recognize. 

SUBPHYLUM  3P0R0Z0A 
Class  Telosporidia 

All  the  members  of  this  class  are  parasitic,  but  only 
a  few  have  been  observed  in  man,  and  only  one  genus, 
Plasmodium,  is  of  much  importance  in  human  pathol- 
ogy. Propagation  is  by  means  of  spores,  and  sporula- 
tion  ends  the  life  of  the  individual.  In  some  species 
there  is  an  alternation  of  generations,  in  one  of  which 
sexual  processes  appear.  In  such  cases  the  male  in- 
dividual may  be  provided  with  ilagella.  Otherwise, 
there  are  no  special  organs  of  locomotion. 

1.  Genus  Coccldium. — i.  Coccidium  cuniculi. 
— This  is  a  very  common  parasite  of  the  rabbit  and  has 
been  much  studied;  but  extremely  few  authentic  cases  of 
infection  in  man  have  been  reported.  The  parasite, 
which  when  fully  developed  is  ovoid  in  shape  and  meas- 
ures about  30  to  50  /i  in  length  and  has  a  shell-like 
integument,  develops  within  the  epithelial  cells  of  the 
bile-passages.  Upon  reaching  adult  size  it  divides  into 
a  number  of  spores  or  merozoites  which  enter  other 
epithelial  cells  and  repeat  the  cycle.  A  sexual  cycle 
outside  the  body,  which  suggests  that  of  the  malarial 
parasite  but  does  not  require  an  insect  host,  also  occurs. 
Infection  takes  place  from  ingestion  of  the  resulting 
sporozoites. 

2.  Genus  Plasmodium. — This  genus  includes  the 
malarial  parasites  which  have  already  been  described 
(see  pp.  349-362). 


PHYLUM   PROTOZOA  47 1 

5.  Genus  Babesia. — The  proper  position  of  this 
genus  is  uncertain.  It  is  placed  among  the  flagellates 
by  some.  The  chief  member  is  Babesia  higeminum, 
the  cause  of  Texas  fever  in  cattle.  It  is  a  minute, 
pear-shaped  organism,  lying  in  pairs  within  the  red 
blood-corpuscles.  An  organism  found  in  the  red  blood- 
corpuscles  and  certain  tissue  cells  in  Rocky  Mountain 
spotted  fever  was  at  one  time  placed  in  this  genus  under 
the  name  B.  (or  Piroplasma)  hominis,  but  its  classifi- 
cation is  uncertain. 

SUBPHYLUM  INFUSORIA 
Class  Ciliata 

The  conspicuous  feature  of  this  class  is  the  presence 
of  cilia.  These  are  hair-like  appendages  which  have  a 
regular  to-and-fro  motion,  instead  of  the  irregular  lash- 
ing motion  of  flagella.  They  are  also  shorter  and  more 
numerous  than  flagella.  Most  infusoria  are  of  fixed 
shape  and  contain  two  nuclei.  Contractile  and  food- 
vacuoles  are  also  present.  Encystment  is  common. 
Only  one  species  is  of  medical  interest.  Certain  ciliated 
structures,  which  have  been  described  as  infusoria, 
notably  in  sputum  and  nasal  mucus,  were  probably 
ciliated  body  cells. 

1.  Genus  Balantidium. — i.  Balantidium  coli. — 
This  parasite,  formerly  called  Paramwcium  coli,  is  an 
occasional  inhabitant  of  the  colon  of  man,  where  it 
sometimes  penetrates  into  the  mucous  membrane  and 
produces  a  diarrheal  condition  resembling  amebic 
dysentery.  It  is  an  actively  moving  oval  organism, 
about  6o  to  loo  )u  long  and  50  to  yd  /i  wide,  is  covered  with 
cilia,    and    contains    a   bean-shaped    macronucleus,   a 


472  ANIMAL    PARASITES 

globular  micronucleus,  two  contractile  vacuoles,  and 
variously  sized  granules  (Fig.  163). 

Its  ordinary  habitat  is  the  large  intestine  of  the 
domestic  pig,  where  it  apparently  causes  no  disturbance. 
It  probably  reaches  man  in  the  encysted  condition. 

2.  Balantidium  minutum  resembles  B.  coli  but  is 
smaller,  measuring  20  to  30  by  15  to  20  jw.  It  has 
been  found  a  few  times  in  diarrheal  stools. 


Fig.  163. — Balantidium  coli  (about    X  350)  (after  Eichhorst). 


PHYLUM  PLATYHELMINTHES 

The  old  phylum  Vermidea  has  been  subdivided  into 
three  phyla,  those  which  are  o^  interest  here  being 
the  Platyhelminthes  and  Nemathelminthes,  the  flat 
worms  and  the  round  worms  respectively.  Of  these, 
many  species  are  parasitic  in  man  and  the  higher  ani- 
mals. In  some  cases  man  is  the  regular  host;  in  others 
the  usual  habitat  is  some  one  of  the  animals,  and  the 
occurrence  of  the  worm  in  man  is  more  or  less  acci- 
dental. Such  are  called  incidejital  parasites.  Only 
those  worms  that  are  found  in  man  with  sufficient 
frequency  to  be  of  medical  interest  are  mentioned 
here. 

The  most  important  means  of  clinical  diagnosis  of 


PHYLUM    PLATYHELMINTHES 


473 


infection  by  either  the  flat  worms  or  the  round  worms 
is  the  finding  of  ova.  In  many  cases  the  ova  are  so 
characteristic  that  the  finding  of  a  single  one  will 
establish  the  diagnosis.  In  other  cases  they  must  be 
carefully  studied  and  a  considerable  number  measured. 
While  ova  from  the  same  species  will  naturally  vary 
somewhat,  the  average  size  of  a  dozen  or  more  is  pretty 
constant.  The  measurements  given  here  are  mainly 
those  accepted  by  Stiles  or  Ward. 

PHYLUM  PL/VTYHELMINTHES 
(Flat  Worms) 


Class  Trematoda. — Flukes.     Unsegmented,  leaf-shaped. 


Genus 

Species 

Fasciola. 

F.  hepatica. 

Dicroccelium. 

D.  lanceatum. 

Opisthorchis. 

Op.  felineus. 

Op.  sinensis. 

Fasciolopsis. 

F.  buski. 

Paragonimus. 

P.  westermani. 

Schistosomum. 

S.  haematobium. 

S.  mansoni. 

S.  japonicum. 

Class  Cestoda. — Tapeworms. 

Segmented,  ribbon-shap 

Genus 

Species 

Taenia. 

T.  saginata. 

T.  solium. 

T.  echinococcus. 

Hymenolepis. 

H. nana. 

H.  diminuta. 

Dipylidium. 

D.  caninum. 

Dibothriocephalus. 

D.  latus. 

474  ANIMAL    PARASITES 

Class  Trematoda 

The  trematodes,  commonly  known  as  ''flukes," 
are  flat,  unsegmented,  generally  tongue-  or  leaf-shaped 
worms.  They  are  comparatively  small, .  most  species 
averaging  between  5  and  15  mm.  in  length.  They  pos- 
sess an  incomplete  digestive  tract,  without  anus,  and 
are  provided  with  one  or  more  sucking  disks  by  means 
of  which  they  can  attach  themselves  to  the  host.  Some 
are  also  provided  with  hooklets.     Nearly  all  species  are 


Fig.   164. — Fasciola   hepatica;  about   two-thirds  natural  size  (Mosler 
and  Peiper). 

hermaphroditic,  and  the  eggs  of  nearly  all  are  opercu- 
lated  (provided  with  a  lid),  the  only  important  excep- 
tion being  the  several  species  of  Schistosomum.  De- 
velopment takes  place  by  alternation  of  generations,  the 
intermediate  generation  occurring  in  some  water 
animal:  mollusks,  amphibians,  fishes,  etc.  Trematoda 
infection  is  uncommon  in  this  country. 

1.  Genus  Fasciola. — i.  Fasciola  hepatica. — The 
"liver  fluke"  inhabits  the  bile-ducts  of  numerous  her- 
bivorous animals,  especially  sheep,  where  it  is  an 
important  cause  of  disease.     It  brings  about  obstruc- 


PHYLUM   PLATYHELMINTHES  475 

tion  of  the  bile-passages,  with  enlargement  and  degener- 
ation of  the  liver — "liver  rot."  A  species  of  snail 
serves  as  intermediate  host.  The  worm  is  leaf-shaped, 
the  average  size  being  about  2.8  by  1.2  cm.  The  an- 
terior end  projects  like  a  beak  (head-cone  3  to  4  mm. 
long)  (Fig.  164).  Ova  appear  in  the  feces.  They  are 
yellowish  brown,  oval,  operculated,  and  measure  about 
130  to  140  by  75  to  90  /i. 

2.  Genus  Dicrocoelium. — i.  Dicrocoelium  lancea- 
tum  is  often  associated  with  the  liver  fluke  in  the  bile-pas- 
sages of  animals,  but  is  neither  so  common  nor  so  widely 
distributed  geographically.  It  has.rarely  been  observed 
in  man.  It  is  smaller  (length  about  i  cm.)  and  more 
elongated.  The  eggs  measure  38  to  45  fi  long  and  22  to 
30  n  wide. 

3.  Genus  Opisthorchis. — i.  Opisthorchis  felineus 
inhabits  the  gall-bladder  and  bile-ducts  of  the  domestic 
cat  and  a  few  other  animals.  Infection  in  man  has 
been  repeatedly  observed  in  Europe,  and  especially  in 
Siberia.  The  body  is  flat,  yellowish-red  in  color,  and 
almost  transparent.  It  measures  8  to  11  by  1.5  to  2 
mm.  The  eggs,  which  are  found  in  the  feces,  are  oval, 
with  a  Well-defined  operculum  at  the  narrower  end, 
and  contain  a  ciliated  embryo  when  deposited.  They, 
measure  about  30  by  11  p.. 

2.  Opisthorchis  sinensis,  like  the  preceding  fluke, 
inhabits  the  gall-bladder  and  bile-ducts  of  domestic 
cats  and  dogs.  It  is,  however,  much  more  frequent  in 
man,  being  a  common  and  important  parasite  in  certain 
parts  of  Japan  and  China.  The  number  present  may 
be  very  great;  over  4000  were  counted  in  one  case. 
The  parasite  resembles  Op.  felineus  in  shape  and  color. 


476  ANIMAL    PARASITES 

It  is  10  to  14  mm.  long  and  2.5  to  4  mm.  broad.  The 
eggs  have  a  sharply  defined  lid  and  measure  25  to  30  by 
15  to  17  fx.  When  they  appear  in  the  feces  they  con- 
tain a  ciliated  embryo.  The  intermediate  host  is  un- 
known. 

4.  Genus  Fasciolopsis. — i.  Fasciolopsis  buski. — 
This  fluke  is  parasitic  in  the  duodenum  of  man,  and  is 
widespread  in  the  East,  notably  in  India,  China,  and 
Japan.  A  few  imported  cases  have  been  reported  in 
this  country.  When  in  considerable  numbers  it  causes 
a  bloody  diarrhea  accompanied  by  high  fever.  The 
usual  length  is  about  30  mm.;  width,  10  to  12  mm.; 
thickness,  1.5  to  4  mm.  The  eggs  are  thin  shelled,  with 
granular  contents,  possess  a  minute  operculum,  and 
measure  about  125  by  75  to  80  /x. 

5.  Genus  Paragonimus. — i.  Paragonimus  wes- 
termannii,  called  the  "lung  fluke,"  is  also  a  common 
parasite  of  man  in  Japan,  China,  and  Korea.  It 
inhabits  the  lung,  causing  the  formation  of  small 
cavities.  Moderate  hemoptysis  is  the  principal  symp- 
tom. Ova  are  readily  found  in  the  sputum  (Fig.  165); 
the  worms  themselves  are  seldom  seen,  except  post- 
mortem. The  worms  are  faintly  reddish-brown  in  color, 
egg  shaped,  with  the  ventral  surface  flattened,  and 
measure  8  to  10  by  4  to  6  mm.  The  ova  are  thin 
shelled,  operculated,  brownish  yellow,  and  measure 
about  87  to  100  by  52  to  66  /x. 

The  larval  stage  occurs  in  several  species  of  fresh- 
water crab  which  are  common  articles  of  food  in 
Japan. 

According  to  Ward,  three  distinct  species  have  been 
confused  under  the  name  P.  westermani:  the  original 


PHYLUM    PLATYHELMINTHES  477 

form,  p.  westermani,  found  in  the  tiger;  the  American 
lung  fluke,  P.  kellicotti,  thus  far  found  only  in  cat,  dog, 
and  hog;  and  the  Asiatic  lung  fluke  of  man,  P.  ringeri, 
described  above. 

6.  Genus  Schistose  mum. — i,  Schistosomum 
hsematobium. — This  trematode,  frequently  called  Bil- 
harzia  hcBmatobia,  is  an  extremely  common  cause  of 


j4K' 


.^^^Mi^- 


ii>.^7Vvi.  ''y- 


m 


'IS- 


^'i^--''^wvi- 


Fig.  165. — Sputum  of  man  containing  eggs  of  the  lung  fluke,  greatly 
enlarged  (after  Manson). 

disease  (bilharziasis  or  Egyptian  hematuria)  in  north- 
ern Africa,  particularly  in  Egypt. 

Unlike  the  other  flukes,  the  sexes  are  separate.  The 
male  is  12  to  14  mm.  long  and  i  mm.  broad.  The  body 
is  flattened  and  the  lateral  edges  curl  ventrally,  form- 
ing a  longitudinal  groove,  in  which  the  female  lies 
(Fig.  166).  The  latter  is  cylindric  in  shape,  about  20 
mm.  long  and  0.25  mm.  in  diameter.  The  eggs  are 
an  elongated  oval,  about  120  to  190  /x  long  and  50  to 


478 


ANIMAL    PARASITES 


73  /x  broad,  yellowish  in  color,  and  slightly  transparent. 
They  possess  no  lid,  such  as  characterizes  the  eggs  of 
most  of  the  trematodes,  but  are  provided  with  a 
thorn-like  spine  which  is  placed  at  one  end  (Fig.  167). 
Within  is  a  ciliated  embryo. 


Fig.   166. — Schistosomum  hamalobium,  male  and  female  (about  X  4), 
with  egg  (about    X  70)  (von  Jaksch). 

In  man  the  worm  lives  in  the  veins,  particularly  the 
portal  vein  and  the  veins  of  the  bladder  and  rectum, 
leading  to  obstruction  and  inflammation.  The  eggs 
penetrate  into  the  tissues  and  are  present  in  abundance 


^:- . 

^^     ^ 

£i^ 

ft  * 

»  « i^ 

, 

■  ^^1 

■  * 

0^^^ 

k  5 

1 

i. 

^^fS^ 

3 

_  ..              -,          -  Qt  ^  .-. 

JM^. 

Fig.  167. — Ova  of  Schistosomum  hcematobium  with  pus  corpuscles  in 
urine   (X  250). 

in  the  mucosa  of  the  bladder  and  rectum.  They  also 
appear  in  the  urine  and,  less  commonly,  in  the  feces. 
A  species  of  snail  serves  as  intermediate  host,  and 
infection  in  man  apparently  takes  place  both  by  mouth 
and  through  the  skin. 


PHYLUM    PLATYHELMINTHES 


479 


2.  Schistosomtim  mansoni.— It  has  long  been  ob- 
served that  schistosomum  eggs  in  the  urine  have  usually 
a  terminal  spine,  while  in  the  feces  the  lateral  spine  is 
more  common.     It  is  now  known   that   the  lateral- 


FiG.  i6S. — Ovii  of  Schistosomum  mansoni:  i.  With  spine  out  of 
focus;  2,  in  a  clump  of  red  blood-cells;  3,  apparently  unfertilized;  4, 
usual  appearance  (X  250). 


spined  egg  is  that  of  a  distinct  species,  to  which  the 
name  Schistosomum  mansoni  has  been  given.  It  is 
found  in  Africa  along  with  Schistosomum  hcematobium, 
but  is  especially  prevalent  in  the  West  Indies  and 
Central  America.     The  adult  worms  closely  resemble 


480  ANIMAL   PARASITES 

the  male  and  female  of  S.  hcematohium.  They  inhabit 
the  rectal  and  portal  veins,  and  ova  appear  in  the 
feces,  where  they  are  very  easily  recognized  from  their 
size  and  the  characteristic  spine  (see  Figs.  168  and  182). 
They  are  light  yellow  in  color,  measure  112  to  162  by 
60  to  70  ju,  and  are  provided  with  a  cleanly  cut,  sharply 
pointed  spine,  which  is  situated  at  the  juncture  of  the 
last  and  third  quarter  of  the  egg  and  is  directed  back- 
ward. Within  the  egg  is  a  ciliated  embryo  (mira- 
cidium)  which  can  be  seen  without  difficulty. 

3.  Schistosomum  japonicmn  resembles  S.  hamato- 
bium  morphologically,  but  both  the  male  and  female  are 
smaller.  The  ova,  which  appear  in  the  feces,  are  ovoid, 
thin  shelled,  and  without  lid  or  spine.  They  average 
83  by  62  fjL  in  size,  and  contain  a  ciliated  embryo.  The 
worm  inhabits  the  portal  and  probably  also  other  veins. 

Class  Cestoda 

The  cestodes,  or  tapeworms,  are  very  common  para- 
sites of  both  man  and  the  animals.  In  the  adult  stage 
they  consist  of  a  linear  series  of  fiat,  rectangular  seg- 
ments (proglottides),  at  one  end  of  which  is  a  smaller 
segment,  the  scolex  or  head,  especially  adapted  by 
means  of  sucking  disks  and  booklets  for  attachment 
to  the  host.  The  series  represents  a  colony,  of  which 
the  scolex  is  ancestor.  The  proglottides  are  sexually 
complete  individuals  (in  most  cases  hermaphroditic) 
which  are  derived  from  the  scolex  by  budding.  With 
the  exception  of  the  immature  segments  near  the 
scolex,  each  contains  a  uterus  filled  with  ova. 

The  large  tapeworms,  Tcenia  saginaia,  T.  solium,  and 


PHYLUM    PLATYHELMINTHES  48 1 

Dibothriocephalus  latus,  are  distinguished  from  one  an- 
other mainly  by  the  structure  of  the  scolex  and  of  the 
uterus.  The  scolex  should  be  studied  with  a  low- 
power  objective  or  a  hand  lens.  The  uterus  is  best 
seen  by  pressing  the  segment  out  between  two  plates 
of  glass. 

All  the  tapeworms  pass  a  larval  stage  in  the  tissues  of 
an  intermediate  host,  which  is  rarely  of  the  same  species 
as  that  which  harbors  the  adult  worm.  Within  the  ova 
which  have  developed  in  the  proglottides  of  the  adult 
worm,  and  which  pass  out  with  the  feces  of  the  host, 
there  develop  embryos,  or  oncospheres,  each  provided 
with  three  pairs  of  horny  booklets.  When  the  egg 
is  taken  into  the  intestines  of  a  suitable  animal,  the 
oncosphere  is  liberated  and  penetrates  to  the  muscles 
or  viscera  and  there,  in  the  case  of  most  of  the  tape- 
worms, forms  a  cyst  in  which  develop  usually  one,  but 
sometimes  many,  scolices,  which  are  identical  with  the 
head  of  the  adult  worm.  When  the  flesh  containing 
this  cystic  stage  is  eaten  without  sufficient  cooking 
to  destroy  the  scolices,  the  latter  attach  themselves 
to  the  intestinal  wall  and  produce  adult  tapeworms 
by  budding.  The  oncosphere  of  some  of  the  tapeworms 
leaves  the  egg  in  the  open  and  exists  for  a  time  as  a 
free-living  larva  before  entering  the  intermediate  host. 

Ordinarily,  only  the  adult  stage  occurs  in  man.  In 
the  case  of  Tcenia  echinococcus  only  the  larval  stage  is 
found.  T.  solium  may  infect  man  in  either  stage, 
although  the  cystic  stage  is  rare. 

Since  the  head,  or  scolex,  is  the  ancestor  from  which 
the  worm  is  formed  in  the  intestine,  it  is  important, 
after  giving  a  vermifuge,  to  make  certain  that  the  head 
31 


482 


ANIMAL   PARASITES 


has  been  passed  with  the  worm.     Should  it  remain,  a 
new  worm  will  develop. 

The  principal  tapeworms  found  in  man  belong  to 
the  genera  Taenia,  Hymenolepis,  and  Dibothriocephalus. 


^IHHimil'jJiJiiirwiiiiiiiJiimi 


QHTPni'ii'iiiiiiiiiiJ 


liiimifniiHiTro 


Fig.  169. — Tcenia  saginata  (Eichhorst) 


1.  Genus  Taenia. — i.  Taenia  saginata  (Fig.  169). — 
This,  the  beef  tapeworm,  is  the  common  tapeworm  of 


Pig.   170. — Head  of  Tcenia  saginata  (Mosler  and  Peiper). 

the  United  States,  and  is  widely  distributed  over  the 
world.  Its  length  is  generally  about  4  to  8  meters. 
The  scolex  is  about  the  size  of  a  large  pin-head  (1.5  to 


PHYLUM   PLATYHELMINTHES  483 

2  mm.  in  diameter),  and  is  surrounded  by  four  sucking 
disks,  but  has  no  booklets  (Fig.  170).  The  neck  is 
about  I  mm,  wide.  The  terminal  segments,  which 
become  detached  and  appear  in  the  feces,  measure 
about  18  to  20  mm.  long  by  4  to  7  mm.  wide.  The 
uterus  extends  along  the  midline  of  the  segment  and 
gives  off  twenty  to  thirty  branches  upon  each  side  (see 
Fig.  180,  i). 

The  larval  stage  is  passed  in  the  muscles  of  various 
animals,  especially  cattle.     It  rarely  or  never  occurs  in 


Fig.  171. — Eggs  of  Tcenia  saginata,  magnifications  lOO,  250,  and  500 
diameters. 

man,  hence  there  is  little  or  no  danger  of  infection  from 
examining  feces. 

The  scolex  is  ingested  with  the  meat,  its  capsule  is 
dissolved  by  the  digestive  juices,  and  it  attaches  itself 
to  the  intestinal  wall  by  means  of  its  suckers.  It  then 
develops  into  the  mature  worm,  which  may  grow  very 
rapidly,  even  as  many  as  thirteen  or  fourteen  segments 
being  formed  in  a  day. 

The  ova  are  present  in  the  feces  of  infected  persons, 
sometimes  in  great  numbers.     When,  however,  segments 


484  ANIMAL    PARASITES 

are  passed,  the  ova  for  the  most  part  remain  within 
them  and  comparatively  few  are  found  free  in  the  feces. 
They  are  spheric  or  ovoid,  yellow  to  brown  in  color, 
and  have  a  thick,  radially  striated  shell  (Fig.  171). 
Within  them  the  sLx  hooklets  of  the  embr}-o  (oncosphere) 
can  usually  be  made  out  as  three  pairs  of  parallel  lines. 
The  size  of  the  ova  varies  from  20  to  30  /x  wide  and  30  to 
40  n  long.  Vegetable  cells,  which  are  generally  present 
in  the  feces,  are  often  mistaken  for  them,  although 
there  is  no  great  resemblance. 


Fig.  172. — Head  of  Tania  solium  (Mosler  and  Peiper). 

2.  Taenia  solium,  the  pork  tapeworm  is  very  rare  in 
this  coimtry.  It  is  usually  much  shorter  than  Tcenia 
sagiimta.  The  scolex  is  about  0.6  to  i  mm.  wide,  is 
surrounded  by  four  sucking  disks,  and  has  a  pro- 
jection, or  rostellum,  with  a  double  row  of  horny  hook- 
lets,  usually  twenty-six  to  twenty-eight  in  number 
(Fig.  172).  The  terminal  segments  measure  about  5  to 
6  by  10  to  12  mm.  The  uterus  has  only  seven  to  four- 
teen branches  on  each  side  (see  Fig.  180,  3). 


PHYLUM   PLATYHELMINTHES  485 

The  cysticercus  stage  occurs  ordinarily  in  the  muscles 
of  the  pig,  but  is  occasionally  seen  in  man,  most  fre- 
quently affecting  the  brain  and  eye  {Cysticercus  cellu- 
Ioscb).  There  is,  therefore,  danger  of  infection  from 
handling  feces. 

The  ova  so  closely  resemble  those  of  Tcenia  saginaia 
as  to  be  practically  indistinguishable.  They  average 
about  31  to  36  M  in  diameter  and  are  usually  spheric. 

3.  Taenia  echinococcus. — The  mature  form  of  this 
tapeworm  inhabits  the  intestines  of  the 
dog  and  wolf.  The  larvae  develop  in 
cattle  and  sheep  ordinarily,  but  are 
sometimes  found  in  man,  where  they 
give  rise  to  echinococcus  or  "hydatid" 
disease.  The  condition  is  unusual  in 
America,  but  is  not  infrequent  in 
Central  Europe  and  is  common  in 
Iceland  and  Australia. 

The  adult  parasite  is  2.5  to  5  mm. 
long  and  consists  of  only  four  segments  echinococcul^^''''^n- 

(Fig.     173).       It    contains     many    ova.   larged    (Mosler  and 

When  the  ova  reach  the  digestive  tract 
of  man  the  embryos  are  set  free  and  find  their  way  to 
the  liver,  lung,  or  other  organ,  where  they  develop  into 
cysts,  thus  losing  their  identity.  The  cysts  may  attain 
the  size  of  a  child's  head.  Other  cysts,  called  ''daughter- 
cysts/'  are  formed  within  these.  The  cyst-wall  is  made 
up  of  two  layers,  from  the  inner  of  which  (the  so-called 
"brood  membrane")  there  develop  larvae  which  are 
identical  with  the  head,  or  scolex,  of  the  mature  para- 
site. These  are  ovoid  structures  0.2  to  0.3  mm.  long. 
Each   has   foiir   lateral    suckers  and  a  rostellum  sur- 


486  ANIMAL   PARASITES 

mounted  by  a  double  circular  row  of  horny  booklets. 
The  rostellum  with  its  booklets  is  frequently  invagi- 
nated  into  the  body. 

Diagnosis  of  echinococcus  disease  depends  upon  de- 
tection of  scolices,  free  booklets  which  have  fallen  off 
from  degenerated  scolices,  or  particles  of  cyst-wall  which 


Fig.   174. — Degenerated  scolex  without  booklets  and  free  booklets  of 
Tarda  echinococcus  in  fluid  from  hepatic  cyst  (X  300). 

are  characteristically  laminated  and  usually  have  curled 
edges.  The  lamination  is  best  seen  at  the  torn  edge  of 
the  membrane.  All  of  these  structures  can  be  found  in 
fluid  withdrawn  from  the  cysts  or,  less  frequently,  in 
the  sputum  or  the  urine,  when  the  disease  involves  the 
lung  or  kidney  (see  Figs.  75, 174, 175).    In  such  material 


PHYLUM   PLATYHELMINTHES  487 

the  scolices  are  usually  much  degenerated  and  many  of 
them  have  entirely  lost  the  hooklets.  The  cysts  are 
sometimes  "barren,"  growing  to  a  considerable  size 
without  producing  scolices. 

The  cyst  fluid  is  clear,  between  1.009  ^^^  i-oi5  in 
specific  gravity,  and  contains  a  notable  amount  of  so- 
dium chlorid,  but  no  albumin.  Recently  diagnosis  of 
echinococcus  disease  has  been  made  by  the  complement 
fixation  method. 


M 

^Mf^ 

i^:\ 

'^'^W 

i^Efi    ^-. 

mi 

^Hr  ^^' 

w^ 

Wn^  ■  ■'"^'^    ■:-' 

Fig.  175. — Portion   of    a    degene-  Fig.      176. — Dwarf 

rated  scolex  of  Tcenia  echinococcus,  tapeworm  {Hymenolepis 
showing  circle  of  hooklets.  From  a  nana)  adults.  From 
hepatic  cyst  (X  250).  photographs.     Natural 

size. 

2.  Genus    Hymenolepis. — i.   Hymenolepis    nana, 

the  dwarf  tapeworm  (Figs.  176  and  177),  is  i  to  4.5  cm. 
in  length  and  0.4  to  0.7  mm.  in  breadth  at  the  widest 
part.  The  head  has  a  rostellum  with  a  crown  of 
24  to  30  hooklets.  There  are  about  150  segments. 
Diagnosis  must,  in  general,  depend  upon  the  dis- 
covery of  ova  in  the  feces  since  the  worms  them- 
selves are  usually  partly  disintegrated  when  they  leave 
the  body  and  are  recognized  with  difficulty.  The  ova 
are  nearly  spheric  and  ■  contain  an  embryo  surrounded 
by  two  distinct  membranous  walls,  between  which  is  a 


488 


ANIMAL   PARASITES 


broad  zone  of  gelatinous  substance  (Fig.  178),     The 
outer  membrane  is  about  40  fj.  in  diameter.     The  inner 


Fig.  177. — Dwarf  tapeworm  {Hymenolepis  nana)  head,  middle 
segments,  and  terminal  segments.  Note  the  protruded  rostellum  and 
the  three  suckers.     From  stained  and  mounted  specimens  (X  30).. 


Fig.  178. — Ova  of  Hymenolepis  nana  in  feces.     The  egg  to  the  right 
was  compressed  by  pressure  upon  the  cover  glass  (X  250  and  X  500). 

averages  28  /x,  and  at  each  pole  has  a  slight  projection 
provided  with  indistinct  filamentous  processes,  which 


PHYLUM    PLATYHELMINTHES  489 

may  lie  between  the  two  membranes  in  such  a  way  as 
sometimes  to  simulate  a  third  membrane.  The  em- 
bryo, of  which  only  the  three  pairs  of  booklets  are 
clearly  seen,  fills  the  space  within  the  inner  wall. 

The  worm  is  common  in  Europe  and  America  and  is 
probably  the  most  common  of  all  the  tapeworms  of 
man  in  the  United  States.  It  is  most  frequent  in  chil- 
dren and  is  generally  present  in  large  numbers,  produc- 
ing considerable  digestive  and  nervous  disturbances. 
The  mode  of  infection  is  unknown. 

A  similar  dwarf  tapeworm  which  is  now  believed  to 
be  identical  with  H.  nana  is  a  very  common  parasite  of 
rats. 

2.  Hymenolepis  diminuta  is  a  common  intestinal 
parasite  of  rats.  A  few  cases  of  infection  in  man  have 
been  reported  in  America.  The  parasite  measures  20 
to  60  cm.  in  length,  is  very  narrow,  and  is  composed  of 
600  to  1300  segments.  The  scolex  lacks  booklets.  The 
ova  resemble  those  of  H.  nana,  but  the  outer  shell  is 
thicker  and  sometimes  radially  striated  and  the  filamen- 
tous processes  between  the  two  membranes  are  lacking. 
The  egg  is  56  to  80  /i  in  diameter  and  the  inner  shell, 
which  contains  a  six-hooked  embryo,  measures  about 
24  by  40  IX. 

3.  Genus  Dipylidium. — i.  DipylidJum  caninum, 
sometimes  called  Tcenia  elliptica,  is  a  very  common 
tapeworm  of  dogs  and  cats.  Its  length  is  15  to  35  cm. 
The  head,  globular  in  shape,  is  armed  with  booklets. 
Terminal  segments  are  shaped  like  cucumber  seeds,  8 
to  II  mm.  long  and  1.5  to  3  mm.  broad.  Ova  are 
spheric,  43  to  50  fx  in  diameter,  and  thin  shelled.  They 
contain  a  six-hooked  embryo,  32  to  36  ;u  in  diameter. 


490  ANIMAL   PARASITES 

The  eggs  are  grouped  in  packets  of  eight  to  fifteen  and 
are  usually  passed  from  the  bowel  within  the  proglottids. 

The  intermediate  host  is  the  flea  or  louse.  Infection 
of  human  beings  is  rare,  and  is  mostly  confined  to  chil- 
dren, who  are  probably  infected  from  getting  lice  or 
fleas  of  dogs  or  cats  into  their  mouths. 

4.  Genus  Dibothriocephalus. — i.  Dibothrioceph- 
alus  latus,  the  fish  tapeworm,  sometimes  reaches  20 
meters  in  length,  although  it  is  generally  not  more  than 
one-half  or  one-third  as  long.  When  several  worms  are 
present,  they  are  much  shorter,  often  only  1.5  or  2  me- 
ters.    The  head  is  a  flattened  ovoid,  about  i  mm.  broad 


Fig.  179. — Head  of  Dibothriocephalus  latus  seen  from  the  narrow  side, 
showing  one  of  the  two  grooves  (x  IS). 

and  1.5  mm.  long.  It  is  unprovided  with  either  suckers 
or  booklets,  but  has  two  longitudinal  grooves  which 
serve  the  same  purpose  (Fig.  179).  The  length  of  the 
segments  is  generally  less  than  their  breadth,  mature 
segments  measuring  about  3  by  10  or  12  mm.  The 
uterus,  which  is  situated  in  the  center  of  the  segment, 
is  roset  shaped  (Fig.  180,  2)  and  brown  or  black  in 
color. 

The  number  of  segments  sometimes  exceeds  3000. 
As  a  rule  they  do  not  appear  in  the  feces  singly,  but  in 
chains  of  considerable  length. 

The  larvae,  which  do  not  form  cysts  but  live  as  worm- 
like structures  2  to  3  cm.  long  (plerocercoids) ,  are  found 


PHYLUM   PLATYHELMINTHES 


491 


in  various  fish,  notably  the  pike,  burbot,  grayUng,  and 
certain  trout.  Infection  of  man  prevails  only  in  regions 
where  these  fish  are  found.  It  is  very  common  in 
Japan  and  in  various  countries  of  Europe,  especially 
Ireland  and  the  Baltic  provinces  of  Russia.  A  number 
of  cases  of  infection  have  been  reported  in  this  country, 
a  few  of  which  were  undoubtedly  acquired  here.     Any 


Fig. 


180. — Segments    of — (i)    Taenia    saginala;    (2)   Dibolhriocephalus 
lalus;  (3)  Taenia  solium,  showing  arrangement  of  uterus. 


locality  in  which  favorable  fish  are  native  becomes  a 
possible  center  of  infection  if  the  worm  is  introduced  by 
infected  immigrants. 

The  ova  are  characteristic.  They  measure  about  45 
by  70  fi,  are  brown  in  color,  and  are  filled  with  small 
spherules.  The  shell  is  thin  and  has  a  small  hinged  lid 
at  one  end.     As  the  eggs  appear  in  the  feces  the  lid  is 


492 


ANIMAL    PARASITES 


not  easily  seen,  but  it  may  be  demonstrated  by  suffi- 
cient pressure  upon  the  cover-glass  to  force  it  open 
(Figs.  i8i,  182).  The  only  other  operculated  eggs  met 
with  in  man  are  those  of  the  fluke- worms. 

Dihothriocephalus  latus  is  interesting  clinically  be- 
cause in  many  cases  it  causes  a  very  severe  grade  of 
anemia,  which  may  be  indistinguishable  from  pernicious 
anemia. 


Fig.   181. — Ova   of    Dihothriocephalus   laliis  (X  250  and  500).     The 
lids  were  forced  open  by  pressure  upon  the  cover-glass. 

PHYLUM  NEMATHELMINTHES 
(Round  Worms) 
Class  Nematoda. — Unsegmented,  cylindric  or  fusiform. 
Genus  Species 

Anguillula.  A.  aceti. 

Ascaris.  A.  lumbricoides. 

A.  canis. 
Oxyuris.  O.  vermicularis. 

Filaria.  F.  bancrofti. 

F.  philippinensis. 
F.  perstans. 
F.  diuma. 
F.  medinensis. 


ypi  i'_m  *^T^\%\    "  'v 


I 


^1^ 

^ 


2, 


Fig.  182.- — Showing  comparative  size  of  ova  found  in  the  feces:  i, 
Trichocephalus  trichiuriis;  2,  Ascaris  lumbricoides;  3,  Necalor  ameri- 
canus,  four-cell  stage;  4,  Schistosomum  mansoni;  5,  Tcenia  saginala; 
6,  Dibolhriocephalus  latus,  the  line  of  the  lid  being  out  of  focus  (X  250). 

493 


494  ANIMAL   PARASITES 

Ancylostoma.  A.  duodenale. 

Necator.  N.  americanus. 

Strongyloides.  S.  intestinalis. 

Trichinella.  T.  spiralis. 

Trichocephalus.  T.  trichiurus. 

Class  Nematoda 

The  nematodes,  or  round-worms,  are  cylindric  or 
fusiform  worms,  varying  in  length,  according  to  species, 
from  I  mm.  to  40  or  80  cm.  As  a  rule,  the  sexes  are 
separate.  The  male  is  smaller  and  more  slender  than 
the  female.  In  a  few  cases  the  female  is  viviparous;  in 
most  cases  she  deposits  ova  which  are  characteristic, 
so  that  the  finding  of  a  single  egg  may  establish  the 
diagnosis.  Except  in  a  few  instances  the  young  are 
different  from  the  adult,  and  must  pass  a  certain  larval 
stage  of  development  before  again  reaching  a  host. 
An  intermediate  host  is,  however,  necessary  with  only 
a  few  species. 

1.  Genus  Anguillula. — i.  Anguillula  aceti. — 
This  worm,  commonly  called  the  "vinegar  eel," 
is  usually  present  in  vinegar.  A  drop  of  the  vine- 
gar, particularly  of  the  sediment,  will  frequently 
show  great  numbers,  all  in  active  motion:  males, 
about  I  or  1.5  mm.  long;  females,  somewhat  larger 
and  frequently  containing  several  coiled  embryos; 
and  young,  of  all  sizes  up  to  the  adult  (see  Fig.  76). 

The  vinegar  eel  is  never  parasitic,  but  is  occasion- 
ally met  with  as  a  contamination  in  the  urine  (see 
p.  237),  and  has  there  been  mistaken  for  the  larva 
of  filaria  or  strongyloides. 


PHYLUM   NEMATHELMINTHES  495 

2.   Genus    Ascaris. — i.    Ascaris     Lumbricoides. — 

The  female  is  20  to  40  cm.  long  and  about  5  mm.  thick 
(Fig.  183);  the  male,  15  to  17  cm.  long  and  3  mm.  thick. 
They  taper  to  a  blunt  point  anteriorly  and  posteriorly. 
At  the  anterior  end  are  three  small  lips  which  can  easily 
be  seen  with  a  hand  lens. 
Their  color  is  reddish  or 
brown.  They  are  the  com- 
mon "round-worms"  so  fre- 
quently found  in  children. 
Their  habitat  is  the  small 
intestine.  Usually  several 
individuals  are  present  and 
sometimes  many. 

The  diagnosis  is  made  by 
detection  of  the  worms  or 
ova  in  the  feces.  The  latter 
are  generally  numerous  and 
are  easily  recognized. 
They  are  elliptic,  measuring 
about  50  by  65  to  80  /x,  and 
have  an  unsegmented  pro- 
toplasm. There  is  usually 
a  crescentic  clear  space  at 

,  11,  .1  Fig.    183. — Ascaris    lumbricoides 

each  pole    between  the   con-       (female)  (Mosler  and  Peiper). 

tents    and    the   shell    (Fig. 

184) .  The  shell  is  thick  and  has  a  roughly  mammillated 
or  sculptured  surface  (Fig.  185).  When  only  females 
are  present  in  the  intestine,  and  occasionally  at  other 
times,  one  finds  unfertilized  eggs.  These  are  generally 
much  more  elongated,  have  a  thinner  and  smoother 


496  ANIMAL    PARASITES 

shell,  have  coarsely  granular  contents,  and  lack  the 
crescentic  clear  spaces. 

The  eggs  do  not  hatch  in  the  intestine  V)f  the  original 
host.  They  pass  out  in  the  feces  and,  after  a  variable 
period,  usually  about  five  weeks,  come  to  contain  an 
embryo  which  remains  within  the  shell  until  ingested 
by  a  new  host.  The  embryo  is  very  resistant  and  may 
remain  alive  within  the  shell  for  years,  even,  according 
to  Morris,  when  preserved  in  2  per  cent,  formalin. 
Upon  reaching  the  intestine  of  the  new  host  it  hatches 


Fig.  184. — Ova  of  Ascaris  lumhricoides  in  fresh  feces  (  X  250). 


out   and   develops  into   the   adult   worm   in   about  a 
month. 

2.  Ascaris  canis  is  the  very  common  "stomach 
worm"  of  cats  and  dogs.  It  is  also  known  as  Ascaris 
mystax  and  Toxocara  canis.  It  is  rare  as  a  human 
parasite.  The  male  is  4  to  9  cm.  long;  the  female,  12  to 
20.  Individuals  from  dogs  are  generally  larger  than 
those  from  cats.  The  egg  is  spheric,  68  to  70  n  in  di- 
ameter, and  has  a  thin  shell  with  comparatively  smooth 
surface. 


PHYLUM   NEMATHELMINTHES  497 

3.  Genus  Oxyuris. — i.  Oxyuris  vermicularis. — 

This  is  the  " thread- worm "  or  ''pin- worm"  which 
matures  in  the  small  intestine  and  cecum  and  in  the 
adult  stage  inhabits  the  colon  and  rectum,  especially  of 


Fig.    185. — Egg  of  Ascaris  lutnbricoides,  surface  view  (x  250). 

young  children.  Its  presence  should  be  suspected  in 
all  unexplained  cases  of  pruritus  ani.  The  female  is 
about  9  to  12  mm.  long;  the  male,  about  3  to  5  mm. 


Fig.   186. — Oxyuris  vermicularis,  male  and  female,  natural   size  (after 

Heller). 

The  worms  are  not  infrequently  found  in  the  feces 
particularly  after  an  enema;  the  ova,  rarely.  The 
latter  are  best  found  by  scraping  the  skin  with  a  dull 
knife  at  the  margin  of  the  anus,  where  they  are  de- 
posited by  the  female,  who  wanders  out  from  the  rec- 


498 


ANIMAL   PARASITES 


turn  for  this  purpose,  thus  producing  the  troublesome 
itching.  They  are  asymmetrically  oval  with  one  flat- 
tened side,  are  about  50  fx  long  by  16  to  25  ju  wide,  have 
a  moderately  thin,  double-contoured  shell,  and  when 
deposited  contain  a  partially  developed  embryo  (Fig. 
187).  The  diagnosis  is  best  made  by  giving  a  purgative 
and  searching  the  stool  for  the  adult  worms  (see  p.  427). 
Infection  takes  place  through  swallowing  the  ova. 
Auto-infection  is  likely  to  occur  in  children;  the  ova 
cling  to  the  fingers  after  scratching  and  are  thus  carried 


Fig.  187. — Eggs  of  Oxyuris  vermicular  is:  a,  freshly  deposited,  with 
tadpole-like  embryo;  h,  twelvehoursafter  deposition,  with  nematode- 
like  embryo  (X  500)  (after  Fantham,  Stephens  and  Theobald). 

to  the  mouth.     Diagnosis  can  sometimes  be  made  by 
finding  the  ova  in  the  dirt  beneath  the  finger-nails. 

4.  Genus  Filaria. — i.  Filaria  bancrofti. — The 
adults  are  thread-like  worms,  the  male  about  4  cm.,  the 
female  about  8  cm.,  long.  They  live  in  pairs  in  the 
lymphatic  channels  and  glands,  especially  those  of  the 
pelvis  and  groin,  and  often  occur  in  such  numbers  as 
to  obstruct  the  flow  of  lymph.  This  is  the  most 
common  cause  of  elephantiasis  and  chyluria.  Infec- 
tion is  very  common  in  tropical  and  sub-tropical  coun- 
tries, where  in  some  regions  as  high  as  50  per  cent,  of 
the  natives  harbor  microfilariae.     Even  as  far  north  as 


PHYLUM    NEMATHELMINTHES  499 

Charleston,  S.  C,  Johnson  has  found  over  19  per  cent, 
of  infection  among  the  poorer  classies.  Of  these  only 
one-fourth  showed  any  symptoms  referable  to  the 
filariae. 

The  female  is  viviparous,  and  produces  vast  numbers 
of  larvae,  which  appear  in  the  circulating  blood.  These 
are  conveniently  called  microfilariae;  the  name  Filaria 
sanguinis  hominis,  which  was  formerly  applied  to  them, 
is  incorrect,  since  they  do  not  constitute  a  species. 
These  larvae  are  slender,  being  about  as  wide  as  a  red 
corpuscle  and  0.2  to  0.4  mm.  long  (see  Fig.  139),  and  are 
very  active,  although,  owing  to  the  fact  that  they  are 
inclosed  in  a  loose  transparent  sheath,  they  do  not  move 
about  from  place  to  place.  They  are  found  in  the 
peripheral  blood  chiefly  at  night,  being  usually  easily 
demonstrable  by  eight  o'clock  and  reaching  their  maxi- 
mum number — which  may  be  enormous — about  2  a.m. 
By  the  concentration  method  given  on  page  363  a  few 
can  usually  be  demonstrated  during  the  day.  In  the 
case  of  a  medical  student  from  Porto  Rico  with  no 
symptoms,  Smith  and  Rivas  found  30  microfilariae  per 
cubic  centimeter  of  blood  at  4  p.  m.  and  6500  per  cubic 
centimeter  at  2  a.m.  If  the  patient  change  his  time 
of  sleeping,  they  will  appear  during  the  day.  Infection 
is  carried  by  a  variety  of  mosquito,  which  acts  as  in- 
termediate host. 

Diagnosis  rests  upon  detection  of  larvae  in  the  blood, 
as  described  on  page  362,  but  the  number  of  larvae 
found  bears  little  relation  to  the  severity  of  the  symp- 
toms, since  the  symptoms  are  largely  mechanical  and 
depend  upon  the  localization  of  the  adults  within  the 
body. 


5CXD  ANIMAL    PARASITES 

The  larvae  are  sometimes  found  in  urine  and  chylous 
fluids  from  the  serous  cavities.  Their  motion  is  then 
usually  less  active  than  when  in  blood.  That  shown 
in  Fig.  i88  was  alive  sixty  hours  after  removal  of  the 
fluid.  Larvae  were  present  in  the  blood  of  the  same 
patient. 

A  number  of  other  filariae  whose  larvae  appear  in  the 
blood  are  known,  some  of  them  only  in  the  larval  stage. 


Fig.  1 88. — ^Larva  of  Filaria  bancrofli  in  chylous  hydrocele  fluid; 
length,  300  n\  width,  8  fi.  The  sheath  is  not  shown.  A  number  of  red 
blood-corpuscles  also  appear  (studied  through  courtesy  of  Dr.  S.  D.  Van 
Meter). 


Among  these  are  Filaria  philippinensis  andF.  persians, 
which  exhibit  no  periodicity,  and  F.  loa,  whose  larvae 
appear  in  the  blood  during  the  day.  The  adult  of  the 
last  named  is  especially  frequent  in  the  orbit  and  be- 
neath the  conjunctiva. 

2.  Filaria  medirensis  the  "guinea- worm,"  is  a 
very  interesting  and  important  worm  of  Africa  and 
southern  Asia.     It  has  been  thought  to  be  the  "fiery 


PHYLUM   NEMATHELMINTHES  501 

serpent"  which  molested  the  Children  of  Israel  in  the 
Wilderness. 

The  larva  probably  enters  the  body  through  the  skin 
or  gastro-intestinal  tract.  It  wanders  about  in  the  sub- 
cutaneous tissues  until  maturity,  producing  slight,  if 
any,  symptoms.  The  male,  which  is  very  rarely  seen, 
is  only  about  4  cm.  long.  It  dies  soon  after  the  female 
is  impregnated.  The  adult  female  is  a  very  slender, 
yellowish  worm,  about  50  to  80  cm.  long,  its  appear- 
ance somewhat  suggesting  a  catgut  suture.  When 
gestation  is  complete  the  greater  part  of  the  female's 
body  consists  of  a  uterus  filled  with  embryos.  The 
female  then  travels  to  the  feet  or  ankles  of  the  host  and 
there  causes  the  formation  of  a  red  nodule  and,  finally, 
an  ulcer,  from  the  center  of  which  her  head  protrudes. 
Through  this  great  numbers  of  larvae  are  discharged 
whenever  it  comes  in  contact  with  water.  Little  dam- 
age is  done  unless  the  worm  is  pulled  out,  when  the 
larvae  are  set  free  in  the  tissues  and  cause  serious 
disturbances. 

When  discharged  the  larvae  seek  out  a  small  crus- 
tacean, Cyclops,  which  serves  as  intermediate  host. 

5.  Ancylostoma  duodenale  and  Necator  ameri- 
canus. — These,  the  Old  and  the  New  World  hookworm, 
respectively,  are  among  the  more  harmful  of  the  animal 
parasites.  They  inhabit  the  small  intestine,  often  in 
great  numbers,  and  commonly  produce  an  anemia  which 
is  often  severe  and  sometimes  fatal.  The  presence  of  a 
few,  however,  may  cause  no  disturbance. 

Ancylostoma  duodenale  is  common  in  southern 
Europe  and  in  Egypt.  The  body  is  cylindric,  reddish 
in  color,  and  the  head  is  bent  sharply.     The  oral  cavity 


502 


ANIMAL    PARASITES 


has  six  hook-like  teeth.  The  female  is  12  to  18  mm. 
long  and  the  tail  is  pointed.  The  male  is  8  to  10  mm. 
long  and  the  posterior  end  is  expanded  into  an  umbrella- 
like pouch,  the  caudal  bursa.  The  eggs  are  oval  and 
have  a  thin,  smooth,  transparent  shell.  As  they  appear 
in  the  feces  the  protoplasm  is  divided  into  2,  4,  8,  or 
more  rounded  segments  (Fig.  189).  They  measure  32 
to  38  by  52  to  61  /x. 


Fig.  189. — Ancylcsloma  duodenale:  a,  Male  (natural  size);  6,  female 
(natural  size);  c,  male  (enlarged);  d,  female  (enlarged) ;  c,  head;/,/,/, 
eggs  (after  v.  Jaksch). 


Necator  americanus  is  very  common  in  subtropical 
America,  including  the  southern  part  of  the  United 
States  and  the  West  Indies.  In  Porto  Rico  90  per  cent, 
of  the  rural  population  is  infected.  Isolated  cases, 
probably  imported,  have  been  seen  in  most  of  the 
Northern  States.  The  American  hook-worm  is  smaller 
than  the  Old  World  variety,  the  male  being  7  to  9  mm. 
long,  the  female  9  to  11  mm.     The  four  ventral  hook- 


PHYLUM   NEMATHELMINTHES  503 

like  teeth  are  replaced  by  chitinous  plates.  There  are 
also  differences  in  the  caudal  bursa  of  the  male,  and  in 
the  situation  of  the  vulva  in  the  female.  The  ova 
(Fig.  190)  resemble  those  of  Ancylostoma  duodenale,  but 
are  larger,  36  to  45  by  64  to  75  ju. 

The  life-history  of  the  two  worms  is  probably  the 
same.  The  ova  pass  out  with  the  feces,  and,  under 
favorable  conditions  of  warmth  and  moisture,  develop 
an  embryo  which  hatches  within  a  few  days.  The  re- 
sulting larvas  pass  through  a  stage  of  development  in 
warm  moist  earth,  growing  to  a  length  of  0.5  to  0.6  mm., 
and  moulting  twice.  They  are  then  ready  to  infect  a 
new  host.  In  some  cases  they  probably  reach  the  host's 
intestine  by  way  of  the  mouth,  with  food  or  water;  but 
the  usual  route  is  probably  that  established  by  Loos. 
When  moist  earth  containing  the  larvae  comes  in  con- 
tact with  the  skin,  they  penetrate  into  the  subcutaneous 
tissues.  This  is  favored  by  retention  of  mud  between 
the  toes  of  those  who  go  barefooted.  When  the  larvae 
are  abundant  a  dermatitis  is  induced  ("ground  itch")- 
From  the  subcutaneous  tissue  they  pass  by  way  of 
lymph-  and  blood-streams  to  the  lungs.  Here  they 
make  their  way  into  the  smaller  bronchi,  are  carried  by 
the  bronchial  mucus  to  the  pharynx,  and  are  swallowed. 
They  thus  ultimately  reach  the  small  intestine,  where 
they  develop  into  mature  worms. 

The  diagnosis  of  hookworm  infection,  which  is  assum- 
ing increasing  importance  in  this  country,  must  rest 
upon  detection  of  ova  in  the  feces.  The  worms  them- 
selves seldom  appear  except  after  thymol  and  a  ca- 
thartic. A  small  portion  of  the  feces,  diluted  with  water 
if  necessary,  is  placed  upon  a  slide  and  the  larger  par- 


504 


ANIMAL   PARASITES 


tides  removed.  The  material  is  covered  and  searched 
with  a  i6-mm.  objective.  A  higher  power  may  rarely 
be  necessary  to  positively  identify  an  egg,  but  should 
not  be  used  as  a  finder.     The  eggs  (see  Figs.  182,  190) 


Fig.  190. — Ova   of  Necator   americanus  in  feces.     The  egg,  showing 
three  cells,  is  a  lateral  view  of  a  four-cell  stage  (X  250). 


are  nearly  always  typical,  showing  a  thin  but  very  dis- 
tinct shell,  a  clear  zone,  and  a  finely  granular  segmented 
protoplasm.  A  fight  spot,  representing  the  nucleus, 
can  usually  be  made  out  in  each  segment.  After  having 
once  been  seen  the  eggs  are  not  easily  mistaken. 


PHYLUM    NEMATHELMINTHES 


505 


In  heavy  infections  they  may  be  found  in  nearly  every 
microscopic  field;  in  most  cases,  even  when  so  mild  as  to 
cause  no  symptoms,  they  can  be  found  on  the  first  slide 
examined.  It  is  seldom  necessary  to  search  more  than 
half  a  dozen  slides.  From  the  estimate  of  Dock  and 
Bass  it  seems  probable  that  ova  will  average  at  least  one 
to  the  slide  if  ten  or  more  laying  females  are  present  in 
the  intestine.     Very  old  females  may  fail  to  produce 


Fig.   191. — Diagram  showing  the  differences  in  the  mouth  cavities  of 
the  larvae  of  A,  Slrongyloides,  and  B,  Necator. 


eggs.  When  they  are  scarce,  some  method  of  sedi- 
mentirig  the  feces  should  be  tried  (see  p.  443). 

Pepper's  method  of  concentration  is  simple,  but  is  not 
applicable  to  other  ova  than  those  of  the  hookworm. 
It  is  best  first  to  sediment  the  feces.  A  layer  of  the 
diluted  feces  is  placed  on  a  slide  and  allowed  to  remain 
for  some  minutes.  The  slide  is  then  gently  immersed 
in  water.  The  ova,  which  have  settled  to  the  bottom, 
cling  to  the  glass  and  are  not  washed  away  as  is  other 
material.  This  may  be  repeated  several  times  and 
numerous  eggs  collected. 

Hookworm  larvae  are  not  found  in  fresh  feces,  but  may 


5o6 


ANIMAL   PARASITES 


hatch  within  twenty-four  to 
forty-eight  hours  after  stool 
is  passed.  They  are  then 
easily  mistaken  for  the  larvae 
of  Strongyloides  intestinalis ,  but 
can  be  distinguished  by  the 
depth  of  the  mouth  cavity, 
which  is  about  equal  to  the 
diameter  of  the  larvae  at  the 
posterior  end  of  the  cavity. 
In  St.  intestinalis  the  mouth 
is  about  one-half  as  deeo  (see 
Fig.  191). 

6.  Genus  Strongyloides. 
— I.  Strongyloides  intes- 
tinalis.— Infection  with  this 
worm  is  by  no  means  so  rare  in 
this  country  as  the  few  clinical 
reports  would  indicate.  It  is 
apparently  widespread  in  the 
Southern  States  and  is  very 
common  in  subtropical  coun- 
tries, notably  in  Italy  and  in 
southern  China.  It  seems 
probable  that  the  parasite  is 
the  cause  of  "Cochin  China 
diarrhea,"  although  some 
authorities  regard  it  as 
harmless- 


FiG.  192. — A,  Egg  of  Strongyloides  intestinalis  (parasitic  mother 
worm)  found  in  stools  of  case  of  chronic  diarrhea;  B,  Rnabditiform 
larva  of  Strongyloides  intestinalis  from  the  stools.  (William  Sydney 
Thayer,  in  Journal  of  Experimental  Medicine.) 


PHYLUM   NEMATHELMTNTHES  507 

The  adult  female,  which  reproduces  by  partheno- 
genesis and  is  about  2  mm.  long,  inhabits  the  upper 
portion  of  the  small  intestine,  but  neither  it  nor  the 
ova  appear  in  the  stool  unless  an  active  diarrhea  exists. 
Ordinarily  they  hatch  in  the  intestines,  and  when  in- 
fection is  severe  larvae  can  be  found  in  the  feces  in  large 
numbers.  These  are  the  "rhabditiform  larvse,"  which 
measure  450  to  600  /x  by  16  to  20  n  (Figs.  192,  193). 
They  are  actively  motile,  with  a  striking  ''wriggling" 


Fig.   193. — Rhabditiform    larva   of  Slrongyloides  intestinalis  in  feces 

(X  150). 

motion,  and,  when  the  stool  is  solid,  are  best  found  by 
making  a  small  depression  in  the  fecal  mass,  filling  it  with 
water,  and  keeping  in  a  warm  place  (preferably  an  in- 
cubator) for  twelve  to  twenty-four  hours.  The  larvae 
will  collect  in  the  water,  and  can  be  easily  found  by 
transferring  a  drop  to  a  slide  and  examining  with  a 
i6-mm.  objective.  The  inexperienced  worker  should 
make  sure  that  the  worms  move,  or  he  may  be  misled 
by  the  vegetable  hairs  which  are  generally  present  in 


5o8  ANIMAL   PARASITES 

the  feces.     Certain  of  these  hairs  (notably  those  from 
the  skin  of  a  peach)  closely  resemble  small  worms  (see 

p.  438). 

Outside  the  body  the  rhabditiform  larvae  develop 
into  a  free-Hving,  sexually  differentiated  generation. 
The  young  of  this  generation  are  the  more  slender 
"filariform  larvae,"  which  constitute  the  infective  form. 
Direct  transformation  of  rhabditiform  into  filariform 
larvae  also  occurs.  Infection  takes  place  by  ingestion 
or  by  way  of  the  skin. 

7.  Genus  Trichinella. — i.  Trichinella  spiralis. — 
This  is  a  very  small  worm — adult  males,  1.5  to  1.6  by 
0.04  mm. ;  female,  3  to  4  by  0.06  mm.  Infection  in  man 
occurs  from  eating  of  pork  which  contains  encysted 
larvae  and  is  insufficiently  cooked.  Ordinary  "curing" 
of  pork  does  not  kill  them.  According  to  Winn  heating 
to  55°C.  for  fifteen  minutes  for  each  pound  of  meat  is 
sufficient  to  kill  all  larvae.  Six  days'  refrigeration  at 
o°F.  (— 17. 7°C.)  is  also  effective.  Protection  against  in- 
fection must  be  secured  through  such  measures  as  these; 
meat  inspection  is  of  little  value  unless  every  part  of 
the  carcass  is  examined  and  this,  of  course,  is  imprac- 
ticable. When  the  larvae  reach  the  stomach  the  capsule 
surrounding  them  is  digested  away  and  they  grow  to 
maturity  in  the  small  intestine.  Soon  after  copula- 
tion the  males  die,  and  the  females  penetrate  into  the 
mucous  membrane  where  they  live  for  about  six  weeks, 
giving  birth  to  great  numbers  of  young,  averaging  as 
high  as  1500  from  a  single  female.  The  larvae  migrate 
to  the  striated  muscles,  chiefly  near  the  tendinous 
insertions,  where  they  grow  to  a  length  of  about  0.8 
mm.,  and  finally  become  encysted.     In  this  condition 


PHYLUM   NEMATHELMINTHES 


509 


they  may  remain  alive  and  capable  of  development  for  as 
long  as  twenty-five  years. 

Trichinella  is  widespread  throughout  the  world  and 
is  more  abundant  in  the  United  States  than  the  reported 
cases  of  human  infection  would  lead  one  to  expect.  It 
is  capable  of  living  in  many  animals  but  is  most  common 
in  the  pig  and  the  rat.  Rats  when  once  infected  con- 
tinue the  infection  through  cannibalism.  A  con- 
venient means  of  finding  whether  the  parasite  is  com- 


FlG.  194. —  Trichinella  spiralis  (larvae)  from  head  of  right  gastroc- 
nemius muscle;  seventh  week  of  disease  (i6-mm.  objective;  eye-piece 
4)  (Boston). 


mon  in  a  given  community  is  to  examine  a  series  of 
slaughter-house  rats. 

Excepting  during  the  acute  stage  trichiniasis  is 
generally  accompanied  by  a  marked  eosinophilia. 
The  diagnosis  is  made  by  teasing  out  upon  a  slide  a 
bit  of  muscle,  obtained  in  man  preferably  from  the 
outer  head  of  the  gastrocnemius,  the  insertion  of  the 
deltoid,  or  the  lower  portion  of  the  biceps.  In  the  case 
of  rats  the  diaphragm,  which  is  the  most  likely  site,  is 


5IO  ANIMAL   PARASITES 

pressed  out  between  two  glass  slides.  The  coiled 
larvae  can  easily  be  seen  with  a  i6-mm.  objective  (Fig. 
194).  The  larvae  can  usually  be  found  in  the  spinal 
fluid  and  the  blood  (see  p.  363)  before  they  have 
reached  their  final  resting-place  in  the  muscles.  During 
the  diarrheal  stage  adults  ijiay  be  present  in  the  feces, 
and  are  found  by  diluting  with  water,  decanting 
several  times  and  examining  the  sediment  in  a  very 
thin  layer  in  clean  water  with  a  hand  lens. 

8.  Genus  Trichocephalus. — i.  Trichocephalus 
trichiixrus. — This,  the  "whip-worm,"  is  3.5  to  5  cm. 
long.     Its  anterior  portion  is  slender  and  thread-like, 


A  ^^^       ^■i^   B 
Fig.  195. — Whip-worms  (Trichocephalus  trichiurus) .     A,  females;  B, 
males.     The  posterior  portion  of  the  male  is  usually  coiled  as  is  shown 
at  the  right.     Photographs  of  mounted  specimens.     Natural  size. 

while  the  posterior  portion  is  thicker  (Fig.  195).  It  is 
widely  distributed  geographically,  and  is  one  of  the  most 
common  of  intestinal  parasites  in  this  country.  It  lives 
in  the  large  intestine,  especially  the  cecum,  with  its 
slender  extremity  embedded  in  the  mucous  membrane. 
Whip-worms  do  not,  as  a  rule,  produce  any  symptoms, 
although  gastro-intestinal  disturbances,  nervous  symp- 
toms, and  anemia  have  been  ascribed  to  them.  They, 
as  well  as  many  other  intestinal  parasites,  are  probably 
an  important  factor  in  the  etiology  of  appendicitis, 
typhoid  fever,  and  other  intestinal  infections.  The 
damage  which  they  do  to  the  mucous  membrane  favors 
bacterial  invasion. 


PHYLUM   ARTKROPODA 


5" 


The  number  present  is  usually  small.  The  worms 
themselves  are  rarely  found  in  the  feces.  The  ova,  which 
are  not  often  abundant,  are  easily  recognized  with  the 
i6-mm.  objective.  Although  they  are  comparatively 
small,  their  appearance  is  striking.  They  are  brown, 
ovoid  in  shape,  50  to  54  n  long  by  about  23  n  wide,  and 
have  a  button-like  projection  at  each  end  (Fig.  196). 


,?' 


Fig.  196. — Ova  of  Trichocephalus  trichiurus  in  feces  (X  500). 


PHYLUM  ARTHROPODA 

The  arthropoda  which  are  parasitic  for  man  belong  to 
the  classes  Arachnoidea  and  Insecta.  They  are  nearly 
all  external  parasites,  and  the  reader  is  referred  to  the 
standard  works  upon  diseases  of  the  skin  for  descriptions. 
The  several  species  of  the  louse  {Pediculus  capitis,  P. 
vestimenti,  Phthirius  pubis)  (Figs.  197,  198,  199,  200), 
the  itch  mite  {Sarcoptes  scabiei) ,  and  the  small  organism 
{Demodex  folliculorum)  which  lives  in  the  sebaceous 
glands,  especially  about  the  face,  are  the  most  common 
members  of  this  group. 

A  number  of  flies  may  deposit  their  ova  in  wounds  or 


512 


ANIMAL   PARASITE 


Fig.  197. — Body  louse  (Pedicidus  vestimenii) ,  female  (X  15).  The 
male  is  a  little  smaller  and  the  posterior  end  of  the  abdomen  has  no 
notch.  The  body  louse  is  distinguished  from  the  head  louse  by  its 
larger  side  and  by  the  relative  widths  of  thorax  and  abdomen. 


Fig. 


Fig.   199. 

capitis),   male    (X    15). 


Fig.    198. — Head    louse    {Pedicidus    capitis),   male    (X    15).     The 
female  is  a  little  larger  and  the  posterior  end  of  the  abdomen  is  notched. 
Fig.  199. — Pubic  louse  {Phlhirius  pubis)  (X  15). 


PHYLUM   ARTHROPODA 


513 


in  such  of  the  body  cavities  as  they  can  reach,  and  the 
resulting  maggots  may  cause  intense  irritation.  To 
this  condition  the  general  term  myiasis  is  applied. 
Ova  may  be  swallowed  with  the 
food  and  the  maggots  appear  in 
the  feces.  A  few  cases  in  which 
larvas  have  been  found  in  the 
urinary  passages  have  been  re- 
ported. Probably  most  important 
is  infection  with  the  "screw  worm," 
the  larva  of  Chrysomyia  macellaria, 
which  is  not  rare  in  some  parts  of 
the  United  States,  particularly 
west  of  the  Mississippi.  The  ova 
are  most  commonly  deposited  in 
the  nasal  pas- 
sages, and  the 
larvae,  which 
may  be  present 
in  great  num- 
bers, burrow 
through  the  soft 
parts,  cartilage, 
and  even  bone, 
always  with 
serious  and 
often  with  fatal  results. 

A  few  cases  of  human  infection  with 
Linguatula  serrata  have  been  reported  from  the  Panama 
Canal  Zone  and  from  Brazil,  and  it  may  prove  to  be 
more  common  than  has  been  recognized.  The  parasite 
belongs  to  the  class  Arachnoidea,  which  includes  spiders, 


Fig.  200. — Empty 
egg  of  Pediculus  capi- 
tis on  a  hair  (X  15). 


i'lG.  Jul.  L.ir\-a  of 
Linguatula  serrata  (de 
Faria  and  Travassos). 


514  ANIMAL   PARASITES 

mites,  ticks,  etc.  It  is  not  at  all  rare  in  Europe.  Re- 
lated species  are  common  in  certain  birds  in  North 
America.  Man  may  be  infected  with  either  adult  or 
larval  stages,  the  former  living  in  the  nasal  and  acces- 
sory passages,  the  latter,  encysted,  in  the  internal  organs, 
particularly  the  liver.  The  larvae  may  be  found  in  the 
feces,  and,  because  of  their  serrations,  may  be  mistaken 
for  minute  tapeworms  (Fig.  201).  They  are  white  in 
color  and  measure  about  4  to  6.5  mm.  long  and  0.9  to 
1.5  mm.  broad  at  the  widest  (anterior)  part. 


CHAPTER  VII 

MISCELLANEOUS  EXAMINATIONS 
PUS 

Pus  contains  much  granular  d6bris  and  numerous 
more  or  less  disintegrated  cells,  the  great  majority  being 
polymorphonuclear  leukocytes— so-called  "pus-corpus- 
cles." Eosinophilic  leukocytes  are  common  in  gonor- 
rheal pus  and  in  asthmatic  sputum.  Examination  of 
pus  is  directed  chiefly  to  detection  of  bacteria. 

When  very  few  bacteria  are  present,  culture  methods, 
which  are  outlined  in  Chapter  VIII,  must  be  resorted 
to.  When  considerable  numbers  are  present,  they  can 
be  detected  and  often  identified  in  cover-glass  smears. 
Several  smears  should  be  made,  dried,  and  fixed,  as 
described  on  page  572. 

One  of  these  should  be  stained  with  a  bacterial  stain. 
LofHer's  methylene-blue  and  Pappenheim's  pyronin- 
methyl-green  are  especially  satisfactory  for  this  pur- 
pose. These  stains  are  applied  for  one-half  minute  to 
two  minutes  or  longer,  without  heating;  the  prepara- 
tion is  rinsed  in  water,  dried,  mounted,  and  examined 
with  an  oil-immersion  lens.  Another  smear  should  be 
stained  by  Gram's  method  (see  p.  572).  These  will 
give  information  concerning  all  bacteria  which  may  be 
present,  and  frequently  no  other  procedure  will  be  nec- 
essary for  their  identification. 

515 


5i6 


MISCELLANEOUS    EXAMINATIONS 


The    most    common    pus-producing    organisms    are 
staphylococci  and  streptococci.     They  are  both  cocci, 


Fig.    202. — Staphylococcus   pyogenes   albus   from    an    abscess   of   the 
parotid  gland  (Jakob). 

or  spheres,  their  average  diameter  being  about  0.7  n. 
Staphylococci  are  commonly  grouped  in  clusters,  often 


Fig.  203. — Streptococcus  pyogenes  from  a  case  of  empyema  (Jakob). 

compared  to  bunches  of  grapes  (Fig.  202).     There  are 
several  varieties  which  can  be  distinguished  only  in 


PUS  517 

cultures.  Streptococci  are  arranged  side  by  side,  form- 
ing chains  of  variable  length  (Fig.  203).  Sometimes 
there  are  only  three  or  four  individuals  in  a  chain; 
sometimes  a  chain  is  so  long  as  to  extend  across  several 
microscopic  fields.  Streptococci  are  more  virulent  than 
staphylococci,  and  are  less  commonly  met.  Both  are 
Gram-positive.  Their  cultural  characteristics  are  given 
on  page  581. 

Should  bacteria  resembling  pneumococci  be  found, 
Rosenow's  or  Smith's  method  for  capsules  (see  p.  87) 


Fig.  204. — Diplococcus  pneumonia  from  ulcer  of  cornea  (oil-im- 
mersion objective)  (study  through  courtesy  of  Dr.  C.  A.  Oliver) 
(Boston). 

should  be  tried.  When  these  are  not  available,  cap- 
sules can  usually  be  shown  by  the  method  of  Hiss. 
The  dried  and  fixed  smear  is  covered  with  a  stain  com- 
posed of  5  c.c.  saturated  alcoholic  solution  gentian- 
violet  and  95  c.c.  distilled  water,  and  heated  until  steam 
rises.  The  preparation  is  then  washed  with  20  per 
cent,  solution  of  copper  sulphate,  dried,  and  mounted 
in  Canada  balsam. 

Pneumococci  may  give  rise  to  inflammation  in  many 


5i8 


MISCELLANEOUS   EXAMINATIONS 


locations  (see  p.  85).  When  they  form  short  chains, 
demonstration  of  the  capsule  or  cultural  methods  (see 
p.  581)  may  be  necessary  to  distinguish  them  from 
streptococci. 

If  tuberculosis  be  suspected,  the  smears  should  be 
stained  by  one  of  the  methods  for  the  tubercle  bacillus 
(see  pp.  77  to  79),  or  guinea-pigs  may  be  inoculated. 
The  bacilli  are  generally  difficult  to  find  in  pus,  and 
bacteria-free  pus  would  suggest  tuberculosis. 

Gonococci,  when  typical,  can  usually  be  identified 
with  sufficient  certainty  for  clinical  purposes  in  the 


Fig.  205. — GonocDcci  in  urethral  pus  (x  1500). 


smear  stained  with  Loffler's  methylene-blue  or,  much 
better,  Pappenheim's  pyronin-methyl-green.  They  are 
ovoid  or  coffee-bean-shaped  cocci  which  lie  in  pairs  with 
their  flat  surfaces  together  (Fig.  205) .  They  lie  for  the 
most  part  within  pus-cells,  an  occasional  cell  being 
filled  with  'them,  while  the  surrounding  cells  con- 
tain few  or  none.  Their  intracellular  position  and 
their  appearance  in  clusters  are  very  important  points 
in  their  identification.     While  a  few  are  often  found 


PUS  519 

outside  of  cells,  one  should  hesitate  to  accept  them  as 
gonococci  unless  further  search  reveals  intracellular 
organisms.  It  is  usually  difhcult  to  find  gonococci 
when  many  other  bacteria  are  present,  even  though 
the  pus  is  primarily  of  gonorrheal  origin.  Whenever 
the  identity  of  the  organism  is  at  all  questionable, 
Gram's  method  should  be  tried.  In  rare  instances  it 
may  be  necessary  to  resort  to  cultures.  The  gonococcus 
is  distinguished  by  its  failure  to  grow  upon  ordinary 
media  (see  p.  582). 

Gonococci  are  generally  easily  found  in  pus  from  un- 
treated acute  and  subacute  gonorrheal  inflammations — 
conjunctivitis,  urethritis,  etc. — but  are  found  with  diffi- 
culty in  pus  from  chronic  inflammations  and  abscesses, 
and  in  urinary  sediments. 

In  the  urine  gonococci  are  most  likely  to  be  pres- 
ent in  the  well-known  "gonorrheal  threads"  or  "float- 
ers," which  consist  of  strands  of  mucus  with  entangled 
pus  corpuscles  and  are  suggestive  of  chronic  gonorrhea, 
but  are  by  no  means  diagnostic  of  it.  These  are  fished  out 
with  a  platinum  wire,  spread  upon  slides,  fixed,  and 
stained.  When  floaters  are  absent  it  may  be  necessary  to 
examine  the  sediment  obtained  by  thorough  centrifuga- 
tion.  In  order  to  remove  urea,  which  prevents  proper 
drying  of  the  smear,  the  sediment  may  be  washed  once 
with  water  or  normal  salt.  Smears  should  be  thin  and 
quickly  dried  in  order  that  the  pus-corpuscles  may 
be  as  well  preserved  as  possible.  Very  often  the  pus- 
cells  are  so  shrunken  that  the  contained  gonococci  are 
difficult  to  recognize.  There  is  likewise  difficulty  in 
finding  gonococci  in  vaginal  discharges  unless  com- 
paratively pure  pus  from  the  suspected  lesion  can  be 


520  MISCELLANEOUS    EXAMINATIONS 

obtained;  otherwise  the  organisms  sought  are  to  a 
great  extent  lost  among  the  myriads  of  bacteria  and  the 
epithelial  and  pus-cells  of  the  leukorrheal  discharge. 
Also,  it  should  be  borne  in  mind  that  the  female  geni- 
tals frequently  harbor  a  non-pathogenic  Gram-negati\  e 
diplococcus  which  closely  resembles  the  gonococcus. 

PERITONEAL,  PLEURAL,  AND  PERICARDIAL  FLUIDS 

The  serous  cavities  contain  very  little  fluid  normally, 
but  considerable  quantities  are  frequently  present  as  a 
result  of  pathologic  conditions.  The  pathologic  fluids 
are  classed  as  transudates  and  exudates. 

Transudates  are  non-inflammatory  in  origin.  They 
contain  only  a  few  cells,  and  less  than  2.5  per  cent,  of 
albumin,  and  do  not  coagulate  spontaneously.  The 
specific  gravity  is  below  1.018.  Micro-organisms  are 
seldom  present. 

Exudates  are  of  inflammatory  origin.  They  are 
richer  in  cells  and  albumin,  and  tend  to  coagulate  upon 
standing.  The  specific  gravity  is  above  1.018.  The 
amount  of  albumin  is  estimated  by  Esbach's  method, 
after  diluting  the  fluid,  if  much  albumin  is  present.  A 
mucin-like  substance,  called  serosomucin,  is  likewise 
found  in  exudates.  It  is  detected  by  acidifying  with  a 
few  drops  of  5  per  cent,  acetic  acid,  when  a  white 
cloudy  precipitate  results.  This  reaction  is  very  help- 
ful in  distinguishing  between  transudates  and  exudates, 
although  some  transudates  give  a  slight  turbidity  with 
acetic  acid.  Bacteria  are  generally  present  and  often 
numerous.  When  none  are  found  in  stained  smears  or 
cultures,  tuberculosis  is  to  be  suspected,  and  animal 
inoculation  should  be  resorted  to. 


PERITONEAL,    PLEURAL,    AND    PERICARDIAL   FLUIDS      5 21 

Exudates  are  usually  classed  as  serous,  serofibrinous, 
seropurulent,  purulent,  putrid,  and  hemorrhagic,  which 
terms  require  no  explanation.  In  addition,  chylous  and 
chyloid  exudates  are  occasionally  met,  particularly  in 
the  peritoneal  cavity.  In  the  chylous  form  the  milki- 
ness  is  due  mainly  to  the  presence  of  minute  fat-drop- 
lets, and  is  the  result  of  rupture  of  a  lymph- vessel,  usu- 
ally from  obstruction  of  the  thoracic  duct.  Chyloid 
exudates  are  milky  chiefly  from  proteins  in  suspension, 
or  fine  debris  from  broken-down  cells.  These  exudates 
are  most  frequently  seen  in  carcinoma  and  tuberculosis 
of  the  peritoneum. 

Cytodiagnosis. — This  is  diagnosis  from  a  differen- 
tial count  of  the  cells  in  a  transudate  or  exudate,  par- 
ticularly one  of  pleural  or  peritoneal  origin. 

The  fresh  fluid,  obtained  by  aspiration,  is  centrifugal- 
ized  for  at  least  five  minutes;  the  supernatant  liquid  is 
poured  off;  and  smears  are  made  from  the  sediment  and 
dried  in  the  air.  The  fluid  must  be  very  fresh,  and  the 
smears  must  be  thin  and  quickly  dried,  otherwise  the 
cells  will  be  small  and  shrunken  and  hence  difficult  to 
identify.  The  smears  are  then  stained  with  Wright's 
blood-stain  which  has  been  previously  diluted  with  one- 
third  its  volume  of  pure  methyl  alcohol,  mounted,  and 
examined  with  an  oil-immersion  objective. 

Predominance  of  polymorphonuclear  leukocytes  (pus- 
corpuscles)  points  to  an  acute  infectious  process  (Fig. 
206).  These  cells  are  the  neutrophiles  of  the  blood. 
Eosinophiles  and  mast-cells  are  rare.  In  thin  smears 
they  are  easily  recognized,  the  cytoplasmic  granules 
often  staining  characteristically  with  polychrome- 
methylene-blue-eosin   stains.     In   thick  smears,   upon 


522 


MISCELLANEOUS   EXAMINATIONS 


Fig.  206. — Cytodiagnosis.  Polymorphonuclear  leukocytes  and 
swollen  endothelial  cells  from  acute  infectious  non-tuberculous  pleuri- 
tis  (Percy  Musgrave;  photo  by  L.  S.  Brown). 


Fig.  207. — Cytodiagnosis.     Lymphoid  cells  from  pleural  fluid;  case  of 
tuberculous  pleuritis  (Percy  Musgrave;  photo  by  L.  S.  Brown). 


PERITONEAL,    PLEURAL,    AND    PERICARDIAL   FLUIDS     523 

'the  Other  hand,  they  are  often  small  and  shrunken,  and 
may  be  identified  with  diflSculty,  being  easily  mistaken 
for  lymphocytes. 

A  large  number,  or  even  a  preponderance,  of  eosino- 
philes  is  occasionally  seen  in  the  effusions  following 
artificial  pneumothorax  and  in  those  of  early  tuberculous 
pleuritis.  In  the  latter  case  the  eosinophiles  gradually 
give  place  to  lymphocytes  as  the  disease  progresses. 


F  i    .    .     •-  ,    'niiaKnosis.      Mesothelial      cells     from     transudate     or 

mechanical  effusion  (Percy  Musgrave;  photo  by  L.  S.  Brown). 

Predominance  of  lymphocytes  (Fig.  207)  generally  sig- 
nifies tuberculosis.  They  are  the  same  as  found  in  the 
blood.  The  cytoplasm  is  usually  scanty,  is  often 
ragged,  and  sometimes  is  apparently  absent  entirely. 
Tuberculous  pleurisy  due  to  direct  extension  from  the 
lung  may  give  excess  of  polymorphonuclears  owing  to 
mixed  infection. 

Predominance  of  mesothelial  cells,  few  cells  of  any 
kind  being  present,  indicates  a  transudate  (Fig.  208). 
These  cells  are  large,  with  relatively  abundant  cyto- 


524  MISCELLANEOUS    EXAMINATIONS 

plasm,  and  contain  one,  sometimes  two,  round  or  oval, 
palely  staining  nuclei.  Mesothelial  cells  generally  pre- 
dominate in  carcinoma,  but  are  accompanied  by  con- 
siderable numbers  of  lymphocytes  and  red  blood-cor- 
puscles. Cancer  cells  cannot  be  recognized  as  such, 
although  the  presence  of  mitotic  figures  would  suggest 
malignant  disease. 

CEREBROSPINAL  FLUID 

Examination  of  the  fluid  obtained  by  lumbar  punc- 
ture has  of  recent  years  become  a  very  important  aid  in 
diagnosis,  particularly  in  syphilitic  conditions  of  the 
nervous  system, 

1.  Macroscopic  Examination. — The  amount  ob- 
tainable varies  from  a  few  drops  to  loo  c.c.  Normally, 
the  fluid  is  clear  and  limpid,  resembling  water.  The 
reaction  is  alkaline.  The  specific  gravity  is  1.003  to 
1.008.  Not  infrequently  it  is  tinged  with  fresh  blood 
from  a  punctured  vessel.  This  should  not  be  confused 
with  the  dull-red  or  brown  color  which  is  seen  in  hemor- 
rhagic conditions  like  intraventricular  and  subdural 
hemorrhage  and  hemorrhagic  meningitis.  When  the 
bleeding  is  extensive  and  recent  it  may  give  the  appear- 
ance of  practically  pure  blood. 

In  purulent  meningitis  the  fluid  may  exhibit  varying 
degrees  of  cloudiness,  from  slight  turbidity  to  almost 
pure  pus.  In  the  less  acute  stage  of  the  epidemic  form 
it  is  sometimes  quite  clear. 

After  standing  for  twelve  to  twenty-four  hours  the 
fluid  will  often  coagulate  This  occurs  especially  in  the 
various  forms  of  meningitis,  rarely  in  non-inflammatory 
conditions.  In  tuberculosis  the  coagulum  is  usually 
very  delicate  and  cobweb-like  and  is  not  easily  seen. 


CEREBROSPINAL   FLUID  525 

2.  Chemical  Examination. — Only  a  fev/  constitu- 
ents are  of  clinical  importance. 

(i)  Globulin.-^Traces  are  present  normally.  A  not- 
able increase  occurs  in  acute  inflammations  and  in 
syphilis  and  parasyphilitic  affections.  The  three  tests 
for  globulin  which  follow  are  positive  in  93  to  95  per 
cent,  of  all  cases  of  paresis,  and  are,  therefore,  an  im- 
portant diagnostic  consideration.  When  acute  inflam- 
mation is  excluded,  they  run  practically  parallel  with 
the  Wassermann  reaction  when  the  latter  is  applied  to 
the  spinal  fluid.  They  must  not  be  applied  to  fluid 
containing  blood,  owing  to  the  presence  of  serum- 
globulin. 

Noguchi's  Butyric  Acid  Test. — In  a  small  test-tube  take 
I  to  2  c.c.  of  the  fluid  and  5  c.c.  of  a  10  per  cent,  solution  of 
butyric  acid  in  normal  salt  solution.  The  original  test 
calls  for  one- tenth  these  quantities,  but  they  are  too  small 
for  convenient  manipulation.  Heat  to  boiling  and  im- 
mediately add  I  c.c.  of  normal  sodium  hydroxid  solution 
and  boil  again  for  a  few  seconds.  A  positive  reaction, 
corresponding  to  a  pathologic  amount  of  globulin,  varies 
from  a  distinct  cloudiness  to  a  heavy  flocculent  precipitate 
which  generally  appears  within  twenty  minutes,  but  may 
be  delayed  for  two  hours.  A  slight  opalescence  miay  be 
seen  in  normal  fluids. 

Ammonium  Sulphate  Test. — Globulin  is  precipitated  by 
strong  solutions  of  ammonium  sulphate.  Ross  and  Jones 
apply  the  test  after  the  manner  of  the  ring  tests  for  albumin 
in  the  urine.  In  a  test-tube  or  horismascope  take  a  few 
cubic  centimeters  of  a  completely  saturated  solution  of 
ammonium  sulphate  and  overlay  with  the  suspected  fluid. 
In  the  presence  of  an  excess  of  globulin,  a  clear-cut,  thin, 
grayish-white  ring  appears  at  the  zone  of  contact  of  the 


526 


MISCELLANEOUS   EXAMINATIONS 


two  fluids  within  five  minutes  to  three  hours.     This  test 
appears  to  be  fully  as  reliable  as  the  butyric  acid  test. 

Pandy's  test  is  said  to  be  more  definite  and  more  sen- 
sitive. The  reagent  consists  of  a  saturated  aqueous  solu- 
tion of  phenol  crystals.  To  i  c.c.  of  the  reagent  add  i 
drop  of  the  cerebrospinal  fluid.  A  bluish-white  cloud  in- 
dicates an  abnormal  amount  of  globulin. 

(2)  Colloidal  Gold  Test. — ^Lange's  colloidal  gold  test, 
introduced  in   191 2  and  now  very  widely  used,  con- 


Di 


utions  of  Spinal 


^ 


lui^ 


mt 


UIM\ 


•-0 


{.m^W  DecQlorizah'on 


I 


Pale  Blue 


4 


\ 


me 


3 


w 


v 


Lilac  or  Purple 


</ 


li 


^ 


M'Uue 


• 


,0' 


Brilliant  Red- Orange 


7 


Fig.  209.— Types  of  reactions  in  colloidal  gold  test:  i,  normal 
cerebrospinal  fluid,  no  reaction;  2,  paretic  type;  3,  luetic  type; 
4,  meningitic  type. 


sists  in  mixing  cerebrospinal  fluid  in  certain  proportions 
with  a  solution  of  colloidal  gold.  Normal  cerebrospinal 
fluid  causes  no  change  in  color.  Fluids  from  cases  of 
syphilis  and  certain  pathologic  conditions  of  the  nervous 
system  induce  changes  in  the  color  of  the  gold  solution 
from  red  to  purple,  deep  blue,  pale  blue,  or  colorless. 
Moreover,  the  dilution  at  which  the  maximum  color 
change  occurs  is  more  or  less  characteristic  of  the  differ- 


CEREBROSPINAL   FLUID  527 

ent  pathological  conditions.  The  typical  "curves"  are 
shown  in  Fig.  209.  The  test  gives  its  most  consistent 
and  valuable  results  in  cases  of  general  paresis.  The 
exact  explanation  of  the  reaction  is  still  uncertain. 

The  test  itself  is  relatively  simple,  and  any  difficulty 
may  be  attributed  to  a  faulty  reagent,  the  preparation 
of  which  is  time-consuming  and  uncertain.  The  reagent 
can  be  purchased  ready  prepared. 

Preparation  of  Reagent. — Lange's  Method  {modified  by 
Miller,  Brush,  Hammers  and  Felton). — It  is  imperative  that 
all  water  used  be  triply  distilled  with  avoidance  of  rubber 
connections  in  the  still,  that  the  beaker  used  for  heating  the 
solution  be  of  Jena  or  Nonsol  glass,  and  that  all  glassware 
be  absolutely  clean.  It  is  recommended  that  the  glass  be 
boiled  in  a  solution  of  Ivory  soap,  brushed  thoroughly  under 
the  tap,  rinsed  well,  soaked  for  one-half  hour  or  longer  in 
hot  bichromate  cleaning  fluid  (see  p.  563),  and  imme- 
diately before  use  rinsed  thoroughly  with  distilled  water 
and  finally  with  triply  distilled  water. 

Heat  slowly  1000  c.c.  triply  distilled  water  in  a  beaker. 
When  the  temperature  reaches  6o°C.  add  10  c.c.  of  a  i 
per  cent,  solution  of  chlorid  of  gold  (Merck's  yellow  crystals 
in  sealed  ampoules)  and  7  c.c.  of  a  fresh  2  per  cent,  solution 
of  potassium  carbonate  (Merck's  "Blue  Label")  using  a 
clean  thermometer  as  a  stirring  rod.  At  8o°C.  slowly  add 
10  drops  of  a  I  per  cent,  solution  of  oxalic  acid  (Merck's 
"Blue  Label"),  stirring  briskly  meanwhile.  At  go^C. 
turn  out  the  fire  and  add  5  c.c.  of  Merck's  formaldehyd, 
"40  per  cent.,  highest  purity,"  drop  by  drop  under  constant 
stirring.  Should  a  pink  color  develop  before  all  the  for- 
maldehyd is  added,  stop  at  once,  for  this  will  slowly  deepen 
to  the  brilliant  orange-red  of  the  finished  solution.  This 
solution  should  be  absolutely  transparent  and  free  from 
any  blue  color;  otherwise  it  is  worthless. 


528  MISCELLANEOUS   EXAMINATIONS 

Before  use  the  solution  must  be  neutralized  with  — 

hydrochloric  acid  or  —  sodium  hydroxid,  as  the  case  may 

be.  The  amount  to  be  added  is  found  by  titrating  a  small 
portion  removed  for  the  purpose,  using  a  i  per  cent,  solu- 
tion of  alizarin  red  in  50  per  cent,  alcohol  as  indicator. 
With  an  acid  reaction  this  indicator  gives  a  lemon-yellow 
color;  with  neutral  reaction,  brownish-red;  with  alkaline, 
red-purple. 

Technic  of  Test. — Arrange  a  series  of  eleven  clean  test- 
tubes.  Place  1.8  c.c.  fresh  sterile  0.4  per  cent,  solution  of 
sodium  chlorid  in  the  first  test-tube  and  i  c.c.  in  each  of 
the  others.  To  the  first  tube  add  0.2  c.c.  of  the  spinal 
fluid,  which  must  be  free  from  any  trace  of  blood.  Mix 
well  by  sucking  the  fluid  up  into  the  pipet  and  expelling 
it,  and  then  transfer  i  c.c.  to  the  second  tube.  Mix  and 
transfer  i  c.c.  to  the  third  tube,  repeating  this  down  the 
row  to  the  tenth  tube  and  discarding  the  last  i-c.c.  portion. 
This  leaves  the  eleventh  tube  with  salt  solution,  only,  to 
serve  as  a  control.  To  each  of  these  eleven  tubes  add  5  c.c. 
of  the  colloidal  gold  solution.  Let  stand  at  room  tem- 
perature for  an  hour  or  longer,  at  the  end  of  which  time, 
in  the  case  of  a  positive  reaction,  the  solution  in  some  of  the 
tubes  will  have  changed  from  red  to  purple,  deep  blue, 
pale  blue,  or  colorless.  In  the  case  of  normal  fluids  no 
change  will  occur.  The  results  are  usually  charted  as 
shown  in  Fig.  209.  -  For  the  purpose  of  brevity  the  colors 
may  be  indicated  by  the  corresponding  numbers,  which  are 
placed  in  the  same  order  as  the  tubes.  Thus  the  "paretic 
reaction"  in  Fig.  209  may  be  expressed  as  5555542100. 

(3)  Mastic  Test. — Because  of  the  many  difficulties  in 
the  way  of  preparing  satisfactory  and  uniform  colloidal 
gold  solutions,  the  mastic  test  has  been  proposed  as  a 


CEREBROSPINAL    FLUID 


529 


satisfactory  substitute  for  the  gold  test.  The  reagent 
is  inexpensive  and  easily  made,  and  the  test  is  easily 
carried  out.  Results  appear  to  parallel  those  obtained 
with  colloidal  gold,  being  almost  uniformly  positive  in 
paresis,  cerebrospinal  syphilis,  and  tabes;  but  there 
does  not  appear  to  be  the  same  opportunity  to  differ- 
entiate various  types  of  reactions  which  the  gold  test 
offers.  The  method  which  follows  is  that  used  by 
Cutting. 


pT 

2 

3 

4 

5  ■  f" 

I 

3     4     5    6 


mill 


Fig.  210. — The  mastic  reaction  in  cerebrospinal  fluid.  A,  from  a 
case  of  dementia  praecox,  negative;  B,  from  a  case  of  paresis,  positive. 
(Courtesy  of  Jas.  A.  Cutting). 

Preparation  of  Solutions. — (a)  Mastic  Solution. — Make  a 
stock  solution  by  completely  dissolving  10  Gm.  of  gum 
mastic,  U.  S.  P.,  in  100  c.c.  of  absolute  alcohol  and  filter. 
To  2  c.c.  of  this  stock  solution  add  18  c.c.  of  absolute 
alcohol,  mix  well,  and  pour  rapidly  into  80  c.c.  of  distilled 
water. 

(6)  Alkaline-saline   Solution. — Make   a    1.25    per    cent, 
solution  of  sodium  chlorid  (C.  P.)  in  distilled  water,  and 
to  each  99  c.c.  of  this  solution  add  i  c.c.  of  a  0.5  per  cent, 
solution  of  potassium  carbonate  in  distilled  water. 
34 


53©  MISCELLANEOUS   EXAMINATIONS 

Technic  of  Test. — Arrange  a  series  of  six  small  test-tubes. 
In  the  first  place  1.5  c.c.  of  the  alkaline-saline  solution  and  in 
each  of  the  others  place  i  c.c.  To  the  first  tube  add  0.5  c.c. 
of  the  spinal  fluid,  which  must  be  completely  free  from 
blood.  Mix  by  sucking  the  fluid  up  into  the  pipet  and 
expelling  it,  and  transfer  i  c.c.  to  the  second  tube.  Again 
mix  and  transfer  i  c.c.  to  the  third  tube  and  continue 
down  the  line  to  the  fifth  tube,  discarding  the  i-c.c.  por- 
tion which  is  removed  from  this  and  leaving  the  sixth  tube 
with  alkaline  saline  solution  alone  to  serve  as  a  control. 
Finally  add  i  c.c.  of  the  mastic  solution  to  each  tube.  Mix 
well  and  set  aside  at  room  temperature  for  twelve  to  twenty- 
four  hours,  or  in  the  incubator  for  six  to  twelve  hours. 
Tubes  in  which  the  reaction  is  complete  will  show  a  heavy 
precipitate  with  clear  supernatant  fluid  (Fig.  210). 

(4)  Sugar. — The  normal  cerebrospinal  fluid  gives  a 
distinct  reaction  with  the  copper  tests  (see  pp*.  162,  163), 
apparently  due  to  glucose,  but  it  is  usually  necessary  to 
use  at  least  twice  as  much  of  the  fluid  as  is  recommended 
for  the  urine.  A  number  of  writers  lay  stress  upon  the 
absence  of  this  reduction  in  meningitis.  From  a  study 
of  a  series  of  cases,  Jacob  finds  that:  (i)  No  reduction 
of  copper  occurs  in  pyogenic  meningitis  (pneumococcus, 
streptococcus,  etc.)  or. in  acute  meningococcic  menin- 
gitis; (2)  reduction  occurs,  but  may  be  diminished  in 
tuberculosis  and  in  the  more  chronic  cases  of  men- 
ingococcic meningitis;  (3)  reduction  is  normal  in 
poliomyelitis. 

(5)  Antimeningococcus-senim  Test. — Vincent  and 
other  French  investigators  have  developed  the  follow- 
ing test,  which  they  believe  to  be  specific  for  epidemic 
cerebrospinal  meningitis : 


CEREBROSPINAL    FLUID  .  53 1 

To  a  few  cubic  centimeters  of  the  spinal  fluid,  which  has 
been  cleared  by  thorough  centrifugation,  are  added  a  few 
drops  of  antimeningococcus  serum.  The  tube,  along  with 
a  control  tube  of  the  untreated  fluid,  is  then  placed  in  an 
incubator  at  52°C.  for  a  few  hours.  A  positive  reaction  con- 
sists in  the  appearance  of  a  white  cloud.  The  test  is  said 
to  be  reliable  even  when  meningococci  cannot  be  found. 
The  serum  must  be  free  from  phenol  and  other  interfering 
substances, 

3.  Microscopic  Examination. — This  consists  in  a 
study  of  the  bacteria,  and  of  the  number  and  kind  of 
cells. 

(i)  Bacteria. — Tubercle  bacilli  can  be  found  in  the 
great  majority  of  cases  of  tuberculous  meningitis.  The 
delicate  coagulum  which  forms  when  the  fluid  is  al- 
lowed to  stand  in  a  cool  place  for  twelve  to  twenty-four 
hours  will  entangle  any  bacilli  which  may  be  present. 
This  clot  may  be  removed,  spread  upon  slides,  and 
stained  by  one  of  the  methods  already  given  (see  pp. 
77  to  79).  If  desired,  the  coagulum  may  be  treated 
with  antiformin  (see  p.  80).  In  case  no  coagulum 
forms,  the  fluid  should  be  thoroughly  centrifugalized 
and  the  sediment  stained,  or,  if  much  protein  be  present, 
it  may  be  coagulated  by  heat,  precipitated  by  the  cen- 
trifuge, and  treated  with  antiformin.  It  may  be  neces- 
sary to  examine  a  considerable  number  of  smears.  In 
doubtful  cases  inoculation  of  guinea-pigs  must  be  re- 
sorted to. 

The  Diplococcus  intracellular  is  meningitidis  is  recog- 
nized as  the  cause  of  epidemic  cerebrospinal  fever,  and 
can  be  detected  in  the  cerebrospinal  fluid  of  most  cases, 
especially  those  which  run  an  acute  course.     Cover- 


532 


MISCELLANEOUS   EXAMINATIONS 


glass  smears  from  the  sediment  should  be  stained  by 
a  simple  bacterial  stain  and  by  Gram's  method.  The 
meningococcus  is  an  intracellular  diplococcus  which 
often  cannot  be  distinguished  from  the  gonococcus  in 
stained  smears  (Fig.  211).  It  also  decolorizes  by  Gram's 
method.  The  presence  of  such  a  diplococcus  in  menin- 
geal exudates  is,  however,  sufficient  for  its  identification 
in  clinical  work. 

Various  organisms  have  been  found  in  other  forms 
of  meningitis — the  pneumococcus  most  frequently,  the 


Fig.  211. —  Meningococci  in  cerebrospinal  fluid  from  a  case  of  epidemic 
spinal  meningitis.    Gram's  method  and  carbol-fuchsin  (X  1500). 

influenza  bacillus  (Fig.  212)  rarely.  When  the  pneumo- 
coccus is  present,  it  is  usually  very  abundant.  In  some 
cases  no  micro-organisms  can  be  detected  even  by  cul- 
ture methods. 

(2)  Cjrtology. — The  fluid  must  be  as  fresh  as  possible  as 
the  cells  tend  to  degenerate.  The  routine  examination 
should  include  both  a  total  and  a  differential  count. 

The  total  number  of  cells  may  be  counted  with  the 
hemacytometer,    but    the    Fuchs-Rosenthal    counting 


CEREBROSPINAL    FLUID  '  533 

chamber  which  is  0.2  mm.  deep  is  more  convenient. 
Unna's  polychrome  methylene-blue  or  a  solution  of 
methyl-violet  or  other  nuclear  dye  is  drawn  into  the 
leukocyte  pipet  to  the  0.5  mark,  and  the  fresh  spinal 
fluid,  which  has  been  well  shaken,  is  drawn  up  to  the 
mark  11.  After  mixing,  a  drop  is  placed  on  the  count- 
ing slide  and  covered.  If  one  is  certain  of  recognizing 
the  cells,  the  dye  may  be  omitted  and  a  small  drop  of 


.  <    ■ 


"  S 


^y^ 


Pig.    212. — Influenza    bacilli    in    spinal    fluid.      Cabc       i     meningitis 

(X   1000). 

the  well  shaken  fluid  placed  directly  upon  the  counting 
slide.  To  reduce  the  error  arising  from  the  small  num- 
ber of  cells  present,  it  is  necessary  to  count  a  large  area 
on  several  slides.  Normally,  the  cells  rarely  exceed  5 
or  7  per  cubic  millimeter;  10  is  perhaps  the  maximum. 
The  differential  count  is  made  as  described  on  page  521. 
Ordinarily,  only  two  kinds  of  cells  are  seen:  lymphocytes 
and  polymorphonuclear  neutrophiles. 
Lymphocytes   predominate   normally.     An   increase 


534  MISCELLANEOUS    EXAMINATIONS 

in  the  total  count,  together  with  predominance  of  lym- 
phocytes (over  70  per  cent.) ,  strongly  suggests  tubercu- 
losis or  syphilitic  disease  of  the  nervous  system,  such  as 
paresis.  It  has  been  observed  in  the  more  chronic  type 
of  epidemic  cerebrospinal  meningitis,  but  not  to  the 
same  extent. 

In  acute  meningitis  the  total  count  is  high  and  poly- 
morphonuclears prevail. 

ANIMAL  INOCULATION 

Inoculation  of  animals  is  one  of  the  most  reliable 
means  of  verifying  the  presence  of  certain  micro-organ- 
isms in  fluids  and  other  pathologic  material,  and  is 
helpful  in  determining  the  species  of  bacteria  which  have 
been  isolated  in  pure  culture. 

Clinically,  it  is  applied  most  frequently  to  demon- 
stration of  the  tubercle  bacillus  when  other  means  have 
failed  or  are  uncertain.  The  guinea-pig  is  the  most 
suitable  animal  for  this  purpose.  When  the  suspected 
material  is  fluid  and  contains  pus,  it  should  be  well 
centrifugalized,  and  i  or  2  c.c.  of  the  sediment  injected, 
by  means  of  a  large  hypodermic  needle,  into  the  peri- 
toneal cavity  or  underneath  the  loose  skin  of  the  groin. 
Fluids  from  which  no  sediment  can  be  obtained  must 
be  injected  directly  into  the  peritoneal  cavity,  since 
at  least  10  c.c.  are  required,  which  is  too  great  an 
amount  to  inject  hypodermically.  Solid  material 
should  be  placed  in  a  pocket  made  by  snipping  the  skin 
of  the  groin  with  scissors,  and  freeing  it  from  the  under- 
lying tissues  for  a  short  distance  around  the  opening. 
When  the  intraperitoneal  method  is  selected,  several 
animals  must  be  inoculated,  since  some  are  likely  to  die 


THE    MOUTH  535 

from  peritonitis  caused  by  other  organisms  before  the 
tubercle  bacillus  has  had  time  to  produce  its  character- 
istic lesions. 

The  animals  should  be  killed  at  the  end  of  six  or  eight 
weeks,  if  they  do  not  die  before  that  time;  and  a  careful 
search  should  be  made  for  the  characteristic  pearl-gray 
or  yellow  tubercles  scattered  over  the  peritoneum  and 
through  the  abdominal  organs,  particularly  the  spleen 
and  liver,  and  for  caseous  inguinal  and  retroperitoneal 
lymph-glands.  The  tubercles  and  portions  of  the 
caseous  glands  should  be  crushed  between  two  slides, 
dried,  and  stained  for  tubercle  bacilli.  The  bacilli  are 
difficult  to  find  in  the  caseous  material. 

It  has  recently  been  shown  that  exposure  of  the 
guinea-pigs  to  strong  a:-ray  for  about  ten  minutes 
so  lowers  their  resistance  that  inoculation  of  tubercu- 
lous material  is  followed  by  recognizable  tuberculosis 
within  about  two  weeks.  Susceptibility  to  other 
bacteria  is  also  increased;  therefore  if  the  material 
contains  pyogenic  organisms  it  should  first  be  treated 
with  antiformin  and  washed  with  water. 

THE  MOUTH 

Micro-organisms  are  always  present  in  large  numbers. 
Among  these  is  Leptothrix  huccalis  (Fig.  213),  which  is 
especially  abundant  in  the  crypts  of  the  tonsils  and  the 
tartar  of  the  teeth.  The  whitish  patches  of  Pharyn- 
gomycosis  leptothrica  are  largely  composed  of  these  fungi. 
They  are  slender,  segmented  threads,  which  generally, 
but  not  always,  stain  violet  with  Lugol's  solution,  and 
are  readily  seen  with  a  4-mm.  objective.  At  times  they 
are  observed  in  the  sputum  and  stomach  fluid.     In  the 


536 


MISCELLANEOUS    EXAMINATIONS 


former  they  might  be  mistaken  for  elastic  fibers;  in  the 
latter,  for  Boas-Oppler  bacilli.  In  either  case,  the  re- 
action with  iodin  will  distinguish  them. 


Fig.  213. — Gingival  deposit  (unstained):  a,  Squamous  epithelial  cells; 
b,  leukocytes;  c,  bacteria;  d,  Leptolhrix  buccalis  (Jakob). 


Fig.  214. — Thrush   fungus    {Endomyces   albicans)  (Kolle  and  Wasser- 

mann). 

The  prevalence  of  endamebae  and  spirochetes  in  the 
mouths  of  normal  persons  and  of  those  suffering  from 


THE    MOUTH  537 

pyorrhea  alveolaris  has  already  been  mentioned  (see 
pp.  457  and  462). 

Thrush  is  a  disease  of  the  mouth  seen  most  often  in 
children,  and  characterized  by  the  presence  of  white 
patches  upon  the  mucous  membrane.  It  is  caused  by 
the  thrush  fungus,  Endomyces  albicans.  When  a  bit 
from  one  of  the  patches  is  pressed  out  between  a  slide 
and  cover  and  examined  with  a  4-mm.  objective,  the 


'7                                   ^                         *- 

/ 

■^                '•Pi 

,       - 

"•  -/r>'o 

\.^>:'c'  ;     '-.- 

,. 

* 

^        •  .                 •.■ 

-— ••     r         \        ••^--     -x              *   . 

^                             '                  '                   V    .      .  "^^^ 

. "  \ " 

/^       .*'      .'■       ..     .  •,-       'r 

Fig.   215. — Bacillus    diphlherice    stained    with    methyl-green;    culture 
from  throat  (X  looo). 

fungus  is  seen  to  consist  of  a  network  of  branching 
segmented  hyphse  with  numerous  spores,  both  within 
the  hyphae  and  in  the  meshes  between  them  (Fig.  214). 
The  meshes  also  contain  leukocytes,  epithelial  cells, 
and  granular  debris. 

Acute  pseudomembranous  inflammations,  which 
occur  chiefly  upon  the  tonsils  and  nasopharynx,  are 
generally  caused  by  the  diphtheria  bacillus,  but  may 


53^  MISCELLANEOUS   EXAMINATIONS 

result  from  streptococcic  infection.  In  many  cases 
diphtheria  bacilli  can  be  demonstrated  in  smears  made 
from  the  membrane  and  stained  with  Loffler's  methy- 
lene-blue  or  2  per  cent,  aqueous  solution  of  methyl- 
green.  They  are  straight  or  curved  rods,  which  vary 
markedly  in  size  and  outline,  and  stain  very  irregularly. 
A  characteristic  form  is  a  palely  tinted  rod  with  several 
deeply  stained  granules  (metachromatic  bodies),  or 
with  one  such  granule  at  each  end  (Fig.  215).  They 
stain  by  Gram's  method.  It  is  generally  necessary, 
and  always  safer,  to  make  a  culture  upon  blood-serum, 
incubate  for  twelve  hours,  and  examine  smears  from 
the  growth. 

Neisser's  stain  has  long  been  the  standard  differential 
stain  for  the  diphtheria  bacillus.  It  colors  the  bodies  of  the 
bacilli  brown  and  the  metachromatic  bodies  blue. 

1.  Make  films  and  fix  as  usual. 

2.  Apply  the  following  solution,  freshly  filtered,  for  about 
one-half  minute: 

Methylene-blue o .  i  Gm.; 

Alcohol  (96  per  cent.) 2.0  c.c; 

Glacial  acetic  acid 5.0  c.c; 

Distilled  water 95  o  c.c. 

3.  Rinse  in  water. 

4.  Apply  a  saturated  aqueous  solution  of  Bismarck 
brown  one-half  minute. 

5.  Rinse,  dry,  and  mount. 

Pender's  Stain. — This  comparatively  new  stain  is  pre- 
ferred by  many  to  Neisser's: 

Toluidin  blue  (Gruebler) 0.02  Gm.; 

Glacial  acetic  acid i  .00  c.c; 

Absolute  alcohol 2 .  00  c.c; 

Distilled  Water  to 100.00  c.c 


THE    MOUTH 


539 


Cover  the  fixed  film  with  the  stain;  turn  the  cover-glass 
over  and  examine  as  a  hanging-drop  preparation.  Diph- 
theria bacilli  are  blue,  with  red  granules. 

Vincent's  angina  is  a  pseudomembranous  and  ulcer- 
ative inflammation  of  mouth  and  pharynx,  which  when 
acute    may   be    mistaken    for    diphtheria,    and    when 


,'  "     ,,x^ 

f  '^'             -^r^        Z^ 

Fig. 


!i6. — SpiyocliccUa    ii,ucnU    from    case     of     ulcerative 
stained  with  gentian-violet  (  X  1200). 


stomatitis 


chronic  is  very  apt  to  be  mistaken  for  syphilis.  Stained 
smears  from  the  ulcers  or  membrane  show  large  numbers 
of  spirochetes  and  ''fusiform  bacilli,"  giving  a  striking 
and  characteristic  picture  (Fig.  216).  Before  making 
the  smears  the  surface  of  the  lesion  should  be  gently 
cleaned  by  swabbing,  otherwise  so  many  miscel- 
laneous bacteria  may  be  present  that  the  char- 
acteristic picture  is  obscured.  The  "bacillus"  is 
spindle-shaped,  more  or  less  pointed  at  the  ends,  and 


540  MISCELLANEOUS    EXAMINATIONS 

about  4  to  8  /i  long.  The  spirillum  is  a  very  slender, 
wavy  thread,  about  lo  to  20  /i  long,  and  stains  feebly. 
Diluted  formalin-gentian-violet  makes  a  satisfactory 
stain.  With  methylene-blue  the  palely  staining  spiril- 
lum may  easily  be  overlooked.  Further  description 
is  given  on  page  462. 

Tuberculous  ulcerations  of  mouth  and  pharynx  can 
generally  be  diagnosed  from  curetings  made  after  care- 
ful cleansing  of  the  surface.  The  curetings  are  well 
rubbed  between  slide  and  cover,  and  the  smears  thus 
made  are  dried,  fixed,  and  stained  for  tubercle  bacilli. 
Since  there  is  much  danger  of  contamination  from 
tuberculous  sputum,  the  presence  of  tubercle  bacilli 
is  significant  only  in  proportion  to  the  thoroughness  with 
which  the  ulcer  was  cleansed.  The  diagnosis  is  certain 
when  the  bacilli  are  found  within  groups  of  cells  which 
have  Jiot  been  dissociated  in  making  the  smears. 

THE  EYE 

Staphylococci,   pneumococci,    and   streptococci   are 

probably  the  most  common  of  the  bacteria  to  be  found 
in  non-specific  conjunctivitis  and  keratitis.  Serpigin- 
ous ulcer  of  the  cornea  is  generally  associated  with  the 
pneumococcus  (see  Fig.  204). 

The  usual  cause  of  acute  infectious  conjunctivitis 
("pink-eye"),  especially  in  cities,  seems  to  be  the  Koch- 
Weeks  bacillus.  This  is  a  minute,  slender  rod,  which 
lies  within  and  between  the  pus-corpuscles  (Fig.  217), 
and  is  negative  to  Gram's  stain.  In  smears  it  cannot 
be  distinguished  from  the  influenza  bacillus,  although 
its  length  is  somewhat  greater. 

The  diplobacillus  of  Morax  and  Axenfeld  gives  rise 
to  an  acute  or  chronic  blepharoconjunctivitis  without 


THE    EYE  541 

follicles  or  membrane,  for  which  zinc  sulphate  seems  to 
be  a  specific.     It  is  widely  distributed  geographically 


Fig.  217. — Conjunctival  secretion  from  acute  contagious  conjunc- 
tivitis; polynuclear  leukocytes  with  the  bacillus  of  Weeks;  P,  phagocyte 
containing  bacillus  of  Weeks  (oil-immersion  objective,  ocular  III) 
(Morax). 


.      Jm    ^   \9    ^^  / 


^ 


Fig.  218. — The  diplobacillus  of  Morax  and  Axenfeld  (from  a  prepara- 
tion by  Dr.  Harold  Giflord). 

and  is  common  in  many  regions.     The  organism  is  a 
short,  thick  diplobacillus,  is  frequently  intracellular,  and 


542  MISCELLANEOUS    EXAMINATIONS 

is  Gram-negative  (Fig.  218).  A  delicate  capsule  can 
sometimes  be  made  out. 

Early  diagnosis  of  gonorrheal  ophthalmia  is  extremely 
important,  and  can  be  made  with  certainty  only  by 
detection  of  gonococci  in  the  discharge.  They  are 
easily  found  in  smears  from  untreated  cases.  After 
treatment  is  begun  they  soon  disappear,  even  though 
the  discharge  continues. 

Pseudomembranous  conjunctivitis  generally  shows 
either  streptococci  or  diphtheria  bacilli.  In  diagnosing 
diphtheric  conjunctivitis,  one  must  .be  on  his  guard 
against  the  Bacillus  xerosis,  which  is  a  frequent  inhabit- 
ant of  the  conjunctival  sac  in  healthy  persons,  and 
which  is  identical  morphologically  with  the  diphtheria 
bacillus.  Hence  the  clinical  picture  is  more  significant 
than  the  microscopic  findings. 

Various  micro-organisms — bacteria,  molds,  proto- 
zoa— have  been  described  in  connection  with  trachoma, 
but  the  more  recent  work  points  to  certain  minute 
intracellular  bodies  as  the  causative  agents.  These 
are  best  seen  in  smears  stained  with  Giemsa's  stain 
and  appear  as  minute  blue  dots  usually  grouped  in 
clusters  in  the  cytoplasm  of  epithelial  cells.  A  red- 
staining  granule  can  be  seen  in  many  of  the  blue  bodies. 
The  nature  of  these  "trachoma  bodies"  is  not  yet 
settled.  They  are  thought  by  many  to  belong  to  the 
chlamydozoa. 

Herbert  has  called  attention  to  the  abundance  of 
eosinophilic  leukocytes  in  the  discharge  of  vernal 
catarrh.  He  regards  their  presence  in  considerable 
numbers  as  very  helpful  in  the  diagnosis  of  this 
disease. 


PARASITIC   DISEASES    OF    THE    SKIN  543 

THE  EAR 

By  far  the  most  frequent  exciting  causes  of  acute 
otitis  media  are  the  pneumococcus  and  the  streptococ- 
cus. The  finding  of  other  bacteria  in  the  discharge 
generally  indicates  a  secondary  infection,  except  in 
cases  complicating  infectious  diseases,  such  as  typhoid 
fever,  diphtheria,  and  influenza.  Discharges  which 
have  continued  for  some  time  are  practically  always 
contaminated  with  the  staphylococcus.  The  presence 
of  the  streptococcus  should  be  a  cause  of  uneasiness, 
since  it  much  more  frequently  leads  to  mastoid  disease 
and  meningitis  than  does  the  pneumococcus.  The 
staphylococcus,  bacillus  of  Friedlander,  colon  bacillus, 
and  Bacillus  pyocyaneus  may  be  met  in  chronic  middle- 
ear  disease. 

In  tuberculous  disease  the  tubercle  bacillus  is  present 
in  the  discharge,  but  its  detection  offers  some  difficulties. 
It  is  rarely  easy  to  find,  and  precautions  must  always  be 
taken  to  exclude  the  smegma  and  other  acid-fast  bacilli 
(see  p.  82),  which  are  especially  liable  to  be  present  in 
the  ear.  Rather  striking  is  the  tendency  of  old  squa- 
mous cells  to  retain  the  red  stain,  and  fragments  of 
such  cells  may  mislead  the  unwary. 

PARASITIC  DISEASES  OF  THE  SKIN 

Favus,  tinea  versicolor,  and  the  various  forms  of 
ringworm  are  caused  by  members  of  the  fungus  group. 
To  demonstrate  them,  a  crust  or  a  hair  from  the  affected 
area  is  softened  with  a  few  drops  of  20  per  cent,  caustic 
soda  solution,  pressed  out  between  a  slide  and  cover, 
and  examined  with  a  4-mm.  objective.     They  consist  of 


544  MISCELLANEOUS   EXAMINATIONS 

a  more  or  less  dense  network  of  hyphai  and  numerous 
round  or  oval  refractive  spores.  The  cuts  in  standard 
works  upon  diseases  of  the  skin  will  aid  in  differentiating 
the  members  of  the  group. 

MILK 

A  large  number  of  analyses  of  human  and  cows'  milk 
are  averaged  by  Holt  as  follows,  Jersey  milk  being  ex- 
cluded because  of  its  excessive  fat: 

Human  Milk  Cows'  Milk 

Normal  variations.    Average,  Average, 

per  cent.  per  cent.  per  cent. 

Fat 3.00  to     5.CX)  4.00  3.50 

Sugar 6.cx)to     7.00  7.00  430 

Proteins i.oo  to     2.25  1.50  4.00 

Salts 0.18  to     0.25  0.20  0.70 

Water 8q  .  82  to  85 .  50  87^30  ^7 .  50 

100.00    100.00  100.00  100.00 

The  reaction  of  human  milk  is  slightly  alkaline;  of 
cows',  neutral  or  slightly  acid.  The  specific  gravity  of 
each  is  about  1.028  to  1.032.  Human  milk  is  sterile 
when  secreted,  but  derives  a  few  bacteria  from  the  lac- 
teal ducts.  Cows'  milk,  as  usually  sold,  contains  large 
numbers  of  bacteria,  the  best  milk  rarely  containing 
fewer  than  10,000  per  cubic  centimeter.  Microscop- 
ically, human  milk  is  a  fairly  homogeneous  emulsion  of 
fat,  and  is  practically  destitute  of  cellular  elements. 
Any  notable  number  of  leukocytes  indicates  infection  of 
the  mammary  gland. 

Chemical  examination  of  milk  is  of  great  value  in  solv- 
ing the  problems  of  infant  feeding.  The  sample  ex- 
amined should  be  the  middle  milk,  or  the  entire  quantity 
from  one  breast.     The  fat  and  protein  can  be  estimated 


MILK 


545 


roughly,  but  accurately  enough  for  many  clinical  pur- 
poses by  means  of  Holt's  apparatus,  which  consists  of 
a  lo-c.c.  cream  gage  and  a  small  hydrometer  (Fig.  219). 
The  cream  gage  is  filled  to  the  o  mark  with  milk. 


A 


f€3 


c.c 

o__io 

2_|_8 
3_L  f 

4_|_6 
5  J|.5 
6_|_4 
7  _|_3 
6_|_2 
9_J_  I 


i2^«i 


Fig.   219. — Holt's  milk-testing  apparatus. 

allowed  to  stand  for  twenty-four  hours  at  room  tem- 
perature, and  the  percentage  of  cream  then  read  off. 
The  percentage'  of  fat  is  three-fifths  that  of  the  cream. 
The  protein  is  then  approximated  from  a  consideration 
of  the  specific  gravity  and  the  percentage  of  fat.     The 

35 


546  MISCELLANEOUS    EX.\MrNATIONS 

salts  and  sugar  verj-  seldom  varj-  sufficiently  to  affect 
the  specific  gra\-ity,  hence  a  high  specific  gra\-it\-  must 
be  due  to  dther  an  increase  of  protein  or  decrease  of  fat, 
or  both,  and  vu:f  versa.  \Mth  normal  specific  gra\'ity 
the  protein  is  high  when  the  fat  is  high,  and  vice  versa. 
The  method  is  not  accurate  with  cows*  milk. 

For   more   accurate   work   the   following   methods, 
a{^Iicable  to  either  human  or  cow's  milk,  are  simple  and 
satisfactory' : 

Fat.—Leffwtaim^Beam  Method.— This, 
is  essentially  the  widely  used  Babcock 
method,  modified  for  the  small  quan- 
tities of  milk  obtainable  from  the  human 
mammary  gland.  The  apparatus  con- 
sists of  a  special  tube  which  fits  the 
aluminum  shield  of  the  medical  centri- 
fuge (Fig.  2  20)  and  a  5-c.c.  pi{)et.  Owing 
to  its  narrow  stem,  the  tube  is  difficult 
to  fill  and  to  clean;  and  for  this  reason 
the  Whitman  modification,  in  which  the 
Fig- 220.— Cen-  Stem  is  removable,  is  to  be  preferred. 
tzibee  tobe  for  Exactlv  s  c.c.  of  the  milk  are  introduced 
mto  the  tube  by  means  of  the  pipet, 
and  I  c.c.  of  a  mixture  of  equal  |>arts  of  concentrated 
hj-drochloric  add  and  amjl-alcohol  is  added  and  well 
mixed.  The  tube  is  filled  to  the  o  mark  with  concen- 
trated sulphuric  add,  adding  a  few  drops  at  a  time  and 
agitating  constantly.  This  is  revolved  in  the  centrifuge 
at  1000  revolutions  a  minute  for  three  minutes,  or  until 
the  fat  has  separated.  The  jjercentage  is  then  read  off 
upon  the  stem,  each  small  division  representing  0.2  per 
cent,  of  fat- 


MILK  54  7 

Proteins. — T.  R.  Boggs'  Modification  of  the  Esbach 
Method. — This  is  applied  as  for  urinary  albumin  (see 
p.  157),  substituting  Boggs'  reagent  for  Esbach's.  The 
reagent  is  prepared  as  follows: 

(i)  Phosphotungstic  add 25  Gm.; 

Distilled  water 125  ex.; 

(2)  Concentrated  hydrochloric  acid 25  ex.; 

Distflled  water 100  ex. 

When  the  phosphotungstic  acid  is  completely  dissolved, 
mix  the  two  solutions.  This  reagent  is  quite  stable  if 
kept  in  a  dark  glass  bottle. 

Before  examination,  the  milk  should  be  diluted  accord- 
ing to  the  probable  amount  of  protein,  and  allowance 
made  in  the  subsequent  reading.  For  human  milk  the 
optimum  dilution  is  i  :  10;  for  cows'  milk,  i  :20.  Dilu- 
tion must  be  accurate. 

Lactose. — The  protein  should  first  be  removed  by 
acidifying  with  acetic  acid,  boiling,  and  filtering.  The 
copper  methods  may  then  be  used  as  for  glucose  in  the 
urine  (see  pp.  166,  167);  but  it  must  be  borne  in  mind 
that  lactose  reduces  copper  more  slowly  than  glucose, 
and  longer  heating  is,  therefore,  required;  and  that 
10  c.c.  of  Fehling's  solution  (or  25  c.c.  of  Benedict's) 
are  equivalent  to  0.0676  Gm.  lactose  (as  compared 
wath  0.05  Gm.  glucose). 

Detection  of  Preservatives. — Formalin  is  the  most 
common  preservative  added  to  cows'  milk,  but  boric 
acid  is  also  used. 

To  detect  formalin,  add  a  few  drops  of  dUute  ferric 
chlorid  solution  to  a  few  cubic  centimeters  of  the  milk, 
and  run  the  mixture  gently  upon  the  surface  of  some 


548  MISCELLANEOUS   EXAMINATIONS 

strong  sulphuric  acid  in  a  test-tube.  If  formaldehyd  be 
present,  a  bright  red  ring  will  appear  at  the  line  of  con- 
tact of  the  fluids.  This  is  not  a  specific  test  for  for- 
maldehyd, but  nothing  else  likely  to  be  added  to  the 
milk  will  give  it. 

To  detect  boric  acid,  Goske's  method  as  used  by  the 
Chicago  Department  of  Health,  is  simple  and  satis- 
factory: Mix  2  c.c.  of  concentrated  hydrochloric  acid 
with  20  c.c.  of  the  milk  and  place  in  a  50-c.c.  beaker. 
In  this  suspend  a  long  strip  of  turmeric  paper  (2  cm. 
wide),  so  that  its  end  reaches  to  the  bottom  of  the 
beaker.  Allow  to  remain  about  half  an  hour.  The 
liquid  will  rise  by  capillarity,  and  if  boric  acid  be  present 
a  red-brown  color  will  appear  at  the  junction  of  the 
moist  and  dry  portions  of  the  paper.  If  this  is  touched 
with  ammonia,  a  bluish-green  slate  color  develops.  A 
rough  idea  of  the  amount  of  boric  acid  may  be  had  by 
comparing  the  depth  of  color  with  that  produced  by 
boric  acid  solutions  of  known  strength. 

SYPHILITIC  MATERIAL 

In  1905  Schaudinn  and  Hoffmann  described  the  occur- 
rence of  a  very  slender,  spiral  micro-organism  in  the 
lesions  of  syphilis.  This  they  named  Spirochata  pallida , 
because  of  its  low  refractive  power  and  the  difficulty 
with  which  it  takes  up  staining  reagents.  The  name 
was  later  changed  to  Treponema  pallidum..  Its  etiologic 
relation  to  syphilis  is  now  universally  admitted.  It  is 
found  in  primary,  secondary,  and  tertiary  lesions,  but 
is  not  present  in  the  latter  in  sufficient  numbers  to  be 
of  value  in  diagnosis 

Treponema  pallidum  is  an  extremely  slender,  spiral, 


syphilitic:  matekial 


549 


motile  thread,  with  pointed  ends.  There  is  a  flagellum 
at  each  end,  but  it  is  not  clearly  seen  in  ordinary  prepa- 
rations. The  organism  varies  considerably  in  length, 
the  average  being  about  7  n,  or  the  diameter  of  a  red 
blood-corpuscle;  and  it  exhibits  three  to  twelve,  some- 
times more,  spiral  curves,  which  are  sharp  and  regular 
and  resemble  the  curves  of  a  corkscrew  (Figs.  158,  221, 
222).     It  is  so  dehcate  that  it  is  difficult  to  see  even 


Fig.  221. —  Treponema  pallidum  (x  looo)  (Leitz  3^2  oil-immersion 
objective  and  Leitz  dark-ground  condenser).  The  parasite  has  the 
same  appearance  as  in  India  ink  preparations. 


in  well-stained  preparations;  a  high  magnification  and 
careful  focusing  are,  therefore,  required.  Upon  ul- 
cerated surfaces  it  is  often  mingled  with  other  spiral 
micro-organisms,  which  adds  to  the  difficulty  of  its 
detection.  The  most  notable  of  these  is  Spirochceta 
refringens,  described  on  page  462. 

Treponema  pallidum  is  most  easily  demonstrated  in 
chancres  and  mucous  patches,  although  the  skin  lesions 
— papules,  pustules,  roseolous  areas — often  contain  large 


55©  MISCELLANEOUS   EXAMINATIONS 

numbers.  Tissue-juice  from  the  deeper  portions  of  the 
lesions  is  the  most  favorable  material  for  examination, 
because  the  organisms  are  commonly  more  abundant 
than  upon  ulcerated  surfaces  and  are  rarely  accompanied 
byother  micro-organisms.  After  cleansing,  the  surface 
is  gently  scraped  with  a  curet  or  rubbed  briskly  with  a 
swab  of  cotton  or  gauze.  In  a  few  moments  serum  will 
exude  and  very  thin  smears  are  then  made  from  it.  In 
transferring  the  serum  from  the  lesion  to  the  slide  or 
cover-glass  it  is  convenient  to  use  a  capillary  pipet. 
The  rubbing  should  not  be  so  vigorous  as  to  bring 
much  blood,  because  the  corpuscles  may  hide  the 
treponema ;  but  a  few-  red  corpuscles  are  an  advantage 
as  an  aid  in  locating  favorable  fields  and  as  a  check 
upon  the  quality  of  the  staining.  Best  fields  are  those 
with  the  clearest  background  and  with  a  few  red  cor- 
puscles, which  must  be  well-stained,  well-preserved, 
and  not  shrunken. 

Staining  Methods. — Giemsa's  stain  (see  p.  3 13)  is  the 

most  widely  used  and  is  perhaps  the  best  (see  Fig.  222). 
It  is  best  purchased  ready  prepared.  Smears  are  fixed  in 
absolute  alcohol  for  fifteen  minutes.  Ten  drops  of  the 
stain  are  added  to  10  c.c.  of  faintly  alkaline  distilled  water 
(i  drop  of  a  I  per  cent,  solution  of  potassium  carbonate 
to  10  c.c.  of  the  water),  and  the  fixed  smear  is  immersed 
on  edge  in  this  diluted  stain  for  one  to  three  hours  or  longer. 
It  is  then  rinsed  in  distilled  water,  dried,  and  mounted. 
More  intense  staining  may  be  obtained  and  the  time  short- 
ened by  conducting  the  process  in  the  incubator.  In 
well-stained  specimens  Treponema  pallidum  is  reddish; 
most  other  micro-organisms,  bluish.  If  desired,  Giemsa's 
stain  may  be  used  as  described  for  blood  (see  p.  313), 
but  the  organisms  do  not  then  stand  out  quite  so  clearly. 


SYPHILITIC   MATERIAL 


.■>o 


It  is  a  waste  of  time  to  search  for  treponemata  in  films 
in  which  the  leukocytes  and  the  red  corpuscles  are  not 
well-stained.  The  nuclei  of  the  former  should  be  dark 
purple;  the  latter  should  be  deep  copper-red  or  salmon- 
colored  when  the  stain  is  used  as  for  blood,  and  deep  slate- 
blue  when  alkali  has  been  added. 


Fig.  222. — Treponema  pallidum,  spirochccta  rejringens,  and  three  red 
blood  corpuscles  in  a  smear  from  a  chancre  (X1200).  From  a  prepa- 
ration stained  with  Giemsa's  stain,  with  alkali,  for  three  hours.  The 
treponemata  were  purplish  red;  refringens,  bluish-purple;  red  corpuscles, 
deep  slate-blue. 

Wright's  blood-stain,  used  in  the  manner  already  de- 
scribed (see  p.  310)  except  that  the  diluted  stain  is  allowed 
to  act  upon  the  film  for  fifteen  minutes,  gives  fair  results. 
Wright  now  recommends  the  following:  In  a  test-tube  mix 
10  c.c.  distilled  water,  i  c.c.  Wright's  stain  and  i  c.c.  of  a 
0.1  per  cent,  solution  of  potassium  carbonate.  Heat  to 
boiling  and  cover  the  preparation  with  the  hot  solution. 


552  MISCELLANEOUS    EXAMINATIONS 

After  three  or  four  minutes  pour  off  the  fluid.     Repeat 
this  twice.     Rinse,  dry  and  mount. 

Silver  Method. — ^The  silver  impregnation  method  has 
long  been  used  for  tissues.  Stein  has  applied  it  to  smears 
as  follows: 

1.  Dry  the  films  in  the  incubator  at  37°C.  for  three  or 
four  hours. 

2.  Immerse  in  lo  per  cent,  silver  nitrate  solution,  in 
diffuse  daylight,  for  some  hours,  until  the  preparation  takes 
on  a  metallic  luster. 

3.  Wash  in  water,  dry,  and  mount. 

The  organisms  are  black  against  a  brownish  background. 

India-ink  Method. — A  small  drop  of  India-ink  of  good 
grade  (Gunther  and  Wagner's  "Chin-Chin  liquid  pearl" 
or  Griibler's  "nach  Burri"  recommended)  is  mixed  on  a 
slide  with  i  or  2  small  drops  of  serum  from  the  suspected 
lesion.  The  mixture  is  then  spread  over  the  slide  and 
allowed  to  dry.  After  drying,  it  is  examined  with  an  oil- 
immersion  lens.  Micro-organisms,  including  Treponema 
pallidum,  appear  clear  white  on  a  brown  or  black  back- 
ground, much  as  they  do  with  the  dark  ground  condenser 
(see  Fig.  221).  If  desired,  the  mixture  of  ink  and  serum 
may  be  covered  with  a  cover-glass  and  examined  in  the 
fresh  state,  the  living  organisms  being  thus  demonstrated. 
Because  of  its  extreme  simplicity  this  method  has  been 
favorably  received.  It  cannot,  however,  be  absolutely 
relied  upon,  since,  as  has  been  pointed  out,  many  India-inks 
contain  wavy  vegetable  fibrils  which  might  easily  mislead 
a  beginner,  and  sometimes,  indeed,  even  an  experienced 
worker.  Instead  of  India-ink,  collargol,  diluted  i:  20  with 
water  and  thoroughly  shaken,  has  been  recommended. 

Dark  ground  illumination  (see  p.  24)  may  be  used  to 
study  the  living  organisms  in  fresh  tissue  juices.  This  offers 
a  satisfactory  means  of  diagnosis,  but  since  the  instrument  is 


SEMEN  553 

expensive  the  practitioner  will  rely  upon  one  or  more  of  the 
staining  methods  just  enumerated. 

Method  of  Oppenheim  and  Sachs. — Very  thin  air-dried 
films  are  stained  for  from  thirty  seconds  to  three  minutes 
with  phenol-gentian-violet  (saturated  alcoholic  solution  of 
gentian-violet,  lo  c.c;  5  per  cent,  phenol,  90  c.c).  Previous 
fixation  is  not  necessary. 

SEMEN 

Absence  of  spermatozoa  is  a  more  common  cause  of 
sterility  than  is  generally  recognized.  In  some  cases 
they  are  present,  but  lose  their  motility  immediately 
after  ejaculation. 

Semen  should  be  kept  warm  until  examined.  When 
it  must  be  transported  any  considerable  distance,  the 
method  suggested  by  Boston  is  convenient:  The  fresh 
semen  is  placed  in  a  small  bottle,  to  the  neck  of  which  a 
string  is  attached.  This  is  then  suspended  from  a  button 
on  the  trousers,  so  that  the  bottle  rests  against  the  skin 
of  the  inguinal  region.  It  may  be  carried  in  this  way 
for  hours.  When  ready  to  examine,  place  a  small  quan- 
tity upon  a  warmed  slide  and  apply  a  cover.  The  sper- 
matozoa are  readily  seen  with  a  4  mm.  objective  (see 
Fig.  74).  Normally,  they  are  abundant  and  in  active 
motion. 

Detection  of  semen  in  stains  upon  clothing,  etc.,  is 
often  important.  The  finding  of  spermatozoa,  after 
soaking  the  stain  for  an  hour  in  normal  salt  solution  or 
dilute  alcohol  and  teasing  in  the  same  fluid,  is  absolute 
proof  that  the  stain  in  question  is  semen  although  it  is 
not  possible  to  distinguish  human  semen  from  that  of 
the  lower  animals  in  this  way.  A  little  eosin  added  to 
the  fluid  will  bring  the  spermatozoa  out  more  clearly. 


554 


MISCELLANEOUS   EXAMINATIONS 


Florence's  Reaction. — The  suspected  material  is  sof- 
tened with  water,  placed  upon  a  slide  w  th  a  few  drops 
of  the  reagent,  and  examined  at  once  with  a  medium 
power  of  the  microscope.  If  the  material  be  semen, 
there  will  be  found  dark-brown  crystals  (Fig.  223)  in  the 
form  of  rhombic  platelets  resembling  hemin  crystals,  or 
of  needles,  often  grouped  in  clusters.     These  crystals  can 


Fig.  223. — Seminal  crystals  (medium  size)  (X  750)  from  a  stain  on 
clothing.  A  single  thread  j^  inch  long  was  used  in  the  test,  the  stain 
being  three  years  and  four  months  old  (Peterson  and  Haines). 

also  be  obtained  from  crushed  insects,  watery  extracts 
of  various  internal  organs,  and  certain  other  substances, 
so  that  they  are  not  absolute  proof  of  the  presence  of 
semen.  Negative  results,  upon  the  other  hand,  are  con- 
clusive, even  when  the  semen  is  many  years'  old. 

The  reagent  consists  of  iodin,  2.54  Gm.;  potassium 
iodid,  1.65  Gm.;  and  distilled  water,  30  c.c. 


DIAGNOSIS    OF   RABIES  555 

DIAGNOSIS  OF  RABIES 

In  view  of  the  brilliant  results  attending  prophylactic 
treatment  by  the  Pasteur  method,  early  diagnosis  of 
rabies  (hydrophobia)  in  animals  which  have  bitten 
persons  is  extremely  important. 

The  most  reliable  means  of  diagnosis  is  the  production 
of  the  disease  in  a  rabbit  by  subdural  or  intracerebral 
injection  of  a  Httle  of  the  filtrate  from  an  emulsion  of 
the  brain  and  medulla  of  the  suspected  animal.  The 
diagnosis  can,  however,  usually  be  quickly  and  easily 
made  by  microscopic  demonstration  of  Negri  bodies. 
Whether  these  bodies  be  protozoan  in  nature  and  the 
cause  of  the  disease,  as  is  held  by  many,  or  whether  they 
be  products  of  the  disease,  it  is  certain  that  their  pres- 
ence is  pathognomonic. 

Negri  bodies  are  sharply  outlined,  round,  oval,  or 
somewhat  irregular  structures  which  vary  in  size,  the 
extremes  being  0.5  and  18  jli.  They  consist  of  a  hyalin- 
like  cytoplasm,  in  which  when  properly  stained  one  or 
more  chromatin  bodies  can  usually  be  seen.  They  are 
situated  chiefly  within  the  .cytoplasm  of  the  large  cells 
of  the  central  nervous  system,  the  favorite  location 
being  the  multipolar  cells  of  the  hippocampus  major 
(Ammon's  horn).  In  many  cases  they  suggest  red 
blood-corpuscles  lying  within  nerve-cells. 

Probably  the  best  clinical  method  of  demonstrating 
Negri  bodies  is  the  impression  method  of  Langdon 
Frothingham,  which  is  carried  out  as  described  below. 

I.  Place  the  dog's  brain^  upon  a  board  about  10  inches 

1  For  Dr.  I-'rothinghatn's  method  of  removing  a  dog's  brain  see 
American  Journal  of  Public  Hygiene  for  February,  1908. 


55 6  MISCELLANEOUS   EXAMINATIONS 

square,  and  divide  into  two  halves  by  cutting  along  the  me- 
dian line  with  scissors. 

2.  From  one  of  the  halves  cut  away  the  cerebellum  and 
open  the  lateral  ventricle,  exposing  the  Ammon's  horn. 

3.  Dissect  out  the  Ammon's  horn  as  cleanly  as  possible. 

4.  Cut  out  a  small  disk  at  right  angles  to  the  long  axis  of 
the  Ammon's  horn,  so  that  it  represents  a  cross-section  of  the 
organ. 

5.  Place  this  disk  upon  the  board  near  the  edge,  with  one 
of  the  cut  surfaces  upward. 

6.  Press  the  surface  of  a  thoroughly  clean  slide  upon  the 
disk  and  lift  it  suddenly.  The  disk  (if  its  exposed  surface 
has  not  been  allowed  to  become  too  dry)  will  cling  to  the 
board,  leaving  only  an  impression  upon  the  slide.  Make 
several  similar  impressions  upon  different  portions  of  the 
slide,  using  somewhat  greater  pressure  each  time.  Im- 
pressions are  also  to  be  made  from  the  cut  surface  of  the 
cerebellum,  since  Negri  bodies  are  sometimes  present  in 
the  Purkinje  cells  when  not  found  in  the  Ammon's  horn. 

7.  Before  the  impressions  dry,  immerse  in  methyl- 
alcohol  for  one-half  to  two  minutes. 

8.  Cover  with  Van  Gieson's  methylene-blue-fuchsin  stain, 
warming  gently  for  one-half  to  two  minutes.  This  stain, 
as  modified  by  Frothingham,  is  as  follows.  It  remains 
effective  for  three  or  four  days: 

Tap-water 20  c.c; 

Saturated  alcoholic  solution  basic  fuchsin i  drop; 

("Fuchsin  f.  Bac,"  Griibler). 
Saturated  aqueous  solution  methylene-blue i  drop. 

("Methylenblau  f.  Bac,  Koch."  Griibler). 

9.  Wash  in  water  and  dry  with  filter-paper.  Examine 
with  a  low  power  to  locate  the  large  cells  in  which  the  bodies 
are  apt  to  be  found,  and  study  these  with  an  oil-immersion 
lens. 


PLATE  XH. 


^: 


Vf- 


m 


Nerve-cells  containing  Negri  bodies. 

Hippocampus  impression  preparation,  dog.  Van  Gieson  stain; 
X  looo.  I,  Negri  bodies;  2,  capillary;  3,  free  red  blood-corpuscles 
(courtesy  of  Langdon  Frothingham). 


DIAGNOSIS    OF    RABIES  557 

The  Negri  bodies  are  stained  a  pale  pink  to  purplish  red, 
and  frequently  contain  small  blue  dots  (Plate  XII).  The 
nerve-cells  are  blue,  and  red  blood-corpuscles  are  colorless 
or  yellowish-copper  colored. 

When  the  work  is  finished,  the  board  with  the  dissected 
brain  is  sterilized  in  the  steam  sterilizer. 

Demonstration  of  Negri  bodies  by  this  method  is  quicker 
and,  possibly,  more  certain  than  by  the  study  of  sections. . 
It  has  the  decided  advantage  over  the  smear  method  that 
the  histologic  structure  is  retained.  One  or  more  of  the 
impressions  generally  shows  the  entire  cell  arrangement 
almost  as  well  as  in  sections,  and  it  is  very  easy  to  locate 
favorable  fields  with  a  i6-mm.  objective. 


CHAPTER  VIII 

BACTERIOLOGIC  METHODS 

Bacteriology  has  become  so  important  a  part  of 
medicine  that  some  knowledge  of  bacteriologic  methods 
is  imperative  for  the  present-day  practitioner.  It  has 
been  the  plan  of  this  book  to  describe  the  various 
bacteria  and  bacteriologic  methods  with  the  subjects  to 
which  they  seemed  to  be  particularly  related.  The 
tubercle  bacillus  and  its  detection,  for  example,  are 
described  in  the  chapters  upon  Sputum  and  Urine; 
blood-cultures  are  discussed  in  the  chapter  upon  Blood. 
There  are,  however,  certain  methods,  notably  the 
preparation  of  media  and  the  study  of  bacteria  by 
cultures,  which  do  not  come  within  the  scope  of  any 
previous  section,  and  an  outline  of  these  is  given  in  the 
present  chapter. 

I.  APPARATUS 

Much  of  the  apparatus  of  the  clinical  laboratory  is 
called    into    use.     Only    the    following    need    special 
mention : 
'I.  Sterilizers. — Two  are  required. 

The  dry,  or  hot-air  sterilizer,  is  a  double- walled  oven 
similar  to  the  detached  ovens  used  with  gas  and  gaso- 
lene stoves.  It  has  a  hole  in  the  top  for  a  perforated 
cork  with  thermometer.  The  oven  of  any  stove,  even 
without  a  thermometer,  will  answer  for  many  purposes. 


APPARATUS  559 

Ordinarily  the  heat  should  be  sufficient  to  slightly 
brown  but  not  char  paper  or  cotton  and  should  be  con- 
tinued for  one-half  to  one  hour. 

The  steam  sterilizer  is  preferably  of  the  Arnold  type, 
opening  either  at  the  top  or  the  side.  An  autoclave, 
which  sterilizes  with  steam  under  pressure,  is  very 
desirable,  but  not  necessary.  An  aluminum  pressure 
cooker  (Fig.  224)  is  a  very  satisfactory  substitute  for 
the  autoclave.     It  costs  about  fifteen  dollars. 


Fig.    224. — Aluminum  pressure   cooker,    an   efficient    and   compara- 
tively inexpensive  substitute  for  an  autoclave. 

2.  Incubator. — This  is  the  most  expensive  piece  of 
apparatus  which  will  be  needed.  It  is  made  of  copper, 
and  has  usually  both  a  water-  and  an  air-jacket  sur- 
rounding the  incubating  chamber.  It  is  provided  with 
thermometer,  thermo-regulator,  and  some  source  of 
heat,  usually  a  Koch  safety  Bunsen  burner.  With  a 
little  ingenuity  one  can  rig  up  a  drawer  or  a  small  box, 
in  which  a  fairly  constant  temperature  can  be  main- 
tained by  means  of  an  electric  light.     The  degree  of 


560  BACTERIOLOGIC    METHODS 

heat  can  be  regulated  by  moving  the  drawer  in  or  out, 
or  holes  can  be  made  in  which  corks  may  be  inserted 
and  removed  as  needed.  A  Thermos  bottle  has  been 
suggested  as  a  temporary  makeshift.  Upon  occasion 
cultures  may  be  kept  warm  by  carrying  them  in  an 
inside  pocket. 

The  gas-heated  copper  incubators  are  now  fast 
being  displaced  by  the  cheaper  and  more  satisfactory 
wooden  incubators  in  which  electricity  is  the  source  of 
heat. 

3.  Culture-tubes  and  Flasks. — For  most  work  ordi- 
nary test-tubes,  125  X  19  mm.  without  flange,  are  satis- 
factory. For  special  purposes  a  few  100  X  13  mm.  and 
150  X  19  mm.  tubes  may  be  needed.  Heavy  tubes, 
which  do  not  easily  break,  can  be  obtained,  and  are  espe- 
cially desirable  when  tubes  are  cleaned  by  an  untrained 
assistant.     The  tubes  are  usually  stored  in  wire  baskets. 

Flasks  of  various  sizes  are  needed.  The  Erlen- 
meyer  type  is  best.  Quart  and  pint  milk  bottles  and 
2 -ounce  wide-mouthed  bottles  will  answer  for  most 
purposes. 

4.  Platinum  Wires. — At  least  two  of  these  are  needed. 
Each  consists  of  a  piece  of  platinum  wire  about  8  cm. 
long,  fixed  in  the  end  of  a  glass  or  metal  rod.  One  is 
made  of  about  22  gage  wire  and  its  end  is  curled  into  a 
loop  2  to  3  mm.  in  diameter.  The  other  wire  is  some- 
what heavier  and  its  tip  is  hammered  flat. 

Lyon  recommends  the  use  of  No.  20  nichrome  wire  as 
nearly  equal  to  platinum  and  very  much  cheaper.  He 
makes  a  handle  of  No.  8,  or  thicker,  aluminum  wire,  sawing 
an  oblique  notch  in  the  end,  inserting  the  nichrome 
wire,  and  hammering  the  aluminum  over  it. 


APPARATUS  561 

5.  Pipets,  etc. — In  addition  to  the  graduated  pipets 
with  which  every  laboratory  is  supplied,  there  are  a 
number  of  forms  which  are  generally  made  from  glass 
tubing  as  needed.  One  of  the  simplest  of  these  is  made 
as  follows:  A  section  of  glass  tubing,  about  12  cm.  long 
and  5  mm.  in  diameter,  is  grasped  at  the  ends,  and  its 
center  is  heated  in  a  concentrated  flame.  A  blast- 
lamp  is  best,  but  a  Bunsen  burner  will  usually  answer, 
particularly   if   fitted   with   a    "wing"    or   "fish-tail" 


Gr?oup>  B 


Fig.  225. — Process  of  making  pipets  (Group  A)  and  Wright's  capsule 
(Group  B).     The  dotted  lines  indicate  where  the  glass  is  to  be  broken. 

attachment.  When  the  glass  is  thoroughly  softened 
it  is  removed  from  the  flame,  and,  with  a  steady, 
but  not  rapid  pull,  is  drawn  out  as  shown  in  Fig.  225. 
The  slender  portion  is  scratched  near  the  middle  with 
a  file  and  is  broken  to  make  two  pipets,  which  are 
then  fitted  with  rubber  nipples.  Two  conditions  are 
essential  to  success:  the  glass  must  be  thoroughly 
softened  and  it  must  be  removed  from  the  flame  before 
beginning  to  pull. 
36 


562  BACTERIOLOGIC    METHODS 

A  nipple  can  be  made  of  a  short  piece  of  rubber 
tubing,  one  end  of  which  is  plugged  with  a  glass  bead. 

This  pipet  has  many  uses  about  the  laboratory. 
When  first  made  it  is  sterile  and  may  be  used  to  trans- 
fer cultures.  With  a  grease-pencil  mark  about  2  cm. 
from  its  tip  (see  Fig.  228),  it  is  useful  for  measuring 
very  small  quantities  of  fluid,  as  in  making  dilutions 
for  the  Widal  test  and  in  counting  bacteria  in  vaccines. 
Mett's  tubes  for  pepsin  estimation  may  be  made  from 
the  capillary  portion.  The  capillary  portion  also 
makes  a  very  satisfactory  blood-lancet  if  the  center  is 
heated  in  a  low  flame  and  the  two  ends  pulled  quickly 
apart. 

Another  useful  device  is  the  Wright  capsule,  which 
is  made  as  shown  in  Fig.  225.  Its  use  is  illustrated  in 
Fig.  230.  After  the  straight  end  is  sealed,  the  curved 
portion  may  be  hooked  over  the  aluminum  tube  of  the 
centrifuge,  and  the  contained  blood  or  other  fluid  sedi- 
mented ;  but  the  speed  should  not  be  so  great  as  to  break 
the  capsule. 

il.  STERILIZATION 

All  apparatus  and  materials  used  in  bacteriologic 
work  must  be  sterilized  before  use. 

Glassware,  metal,  etc.,  are  heated  in  the  hot-air 
sterilizer  at  i5o°C.  for  one  hour,  at  i8o°C.  for  half  an 
hour,  or  at  20o°C  for  five  minutes.  Flasks,  bottles, 
and  tubes  are  plugged  with  cotton  before  heating. 
Petri  dishes  may  be  wrapped  in  paper  in  sets  of  three. 
Pipets  and  glass  or  metal  hypodermic  syringes  are 
placed  in  cotton-stoppered  test-tubes. 

Culture-media  and  other  fluids  must  be  sterilized  by 
steam.     Exposure  in  an  autoclave  to  a  temperature  of 


PREPARATION    OF    CULTURE-TUBES  563 

iio°C.  (6  pounds'  pressure)  for  one-half  hour  or  of 
i2i°C  (about  15  pounds'  pressure)  for  fifteen  minutes  is 
sufficient.  With  the  Arnold  sterilizer,  which  is  more 
commonly  used,  the  intermittent  plan  must  be  adopted, 
since  steam  at  ordinary  pressure  will  not  kill  spores. 
This  consists  in  steaming  for  thirty  to  forty-five  min- 
utes on  three  or  four  successive  days.  Spores  which 
are  not  destroyed  upon  the  first  day  develop  into  the 
vegetative  form  and  are  destroyed  at  the  next  heating. 
Gelatin  media  must  not  be  exposed  to  steam  for  more 
than  twenty  minutes  at  a  time,  and  must  then  be 
removed  from  the  sterilizer  and  cooled  in  cold  water, 
otherwise  the  gelatin  may  lose  its  power  to  solidify. 
Cotton  and  gauze  are  sterilized  by  either  hot  air  or 
steam,  preferably  the  latter. 

IIL  PREPARATION  OF  CULTURE-TUBES 

New  tubes  should  be  washed  in  a  very  dilute  solu- 
tion of  nitric  acid,  rinsed  in  clear  water,  and  allowed  to 
drain  dry. 

Tubes  which  contain  dried  culture-media  are  cleaned 
with  a  test-tube  brush  after  boiling  in  a  strong  solution 
of  washing-soda.  They  are  then  rinsed  successively  in 
clear  water,  acidulated  water,  and  clear  water,  and  al- 
lowed to  drain. 

The  well-known  bichromate  cleaning  fluid  is  very 
valuable  for  cleaning  glassware  of  all  kinds.  It  consists 
of: 

Potassium  bichromate 100  Gm.; 

Concentrated  sulphuric  acid 120  c.c; 

Water 1000  c.c. 


564  BACTERIOLOGIC    METHODS 

Glass-ware  may  be  placed  in  this  solution  for  one  day 
or  longer  and  then  rinsed  thoroughly  and  dried. 

The  tubes  are  now  ready  to  be  plugged  with  raw 
cotton — the  "cotton  batting"  of  the  dry  goods  stores. 
This  is  done  by  pushing  a  wad  of  cotton  into  each  tube 
to  a  depth  of  about  3  cm.  with  a  glass  rod.  The  plugs 
should  fit  snugly,  but  not  too  tightly,  and  should  pro- 
ject from  the  tube  sufficiently  to  be  readily  grasped  by 
the  fingers.  The  tubes  are  next  placed  in  wire  baskets 
and  heated  in  an  oven  for  about  one-half  hour  at  150'^C. 
in  order  to  mold  the  stoppers  to  the  shape  of  the  tubes. 
The  heating  should  not  char  the  cotton,  although  a 
slight  browning  does  no  harm.  The  tubes  are  now 
ready  to  be  filled  with  culture-media. 

IV.  CULTURE-MEDIA 

For  a  careful  study  of  bacteria  a  great  variety  of  cul- 
ture-media is  required,  but  only  a  few — bouillon,  agar 
or  solidified  blood-serum,  and  gelatin — are  much  used 
in  routine  work.  A  great  deal  of  work  can  be  done  with 
a  single  medium,  for  which  purpose  solidified  blood- 
serum  is  probably  best.  The  ordinary  culture-media, 
put  up  in  tubes  ready  for  use,  can  be  purchased  through 
any  pharmacy,  and  some  can  be  obtained  in  powder  or 
tablet  form  ready  to  be  dissolved  in  the  appropriate 
amount  of  water. 

Preparation  of  Culture -media. — 

Beef  Infusion 

Hamburger  steak,  lean 500  Gm.; 

Tap- water 1000  c.c. 

'Mix  well;  let  soak  about  twenty-four  hours  in  an  ice- 


CULTURE-MEDIA  565 

chest,  and  squeeze  through  cheese-cloth.  This  infusion 
is  not  used  by  itself,  but  forms  the  basis  for  various 
media.  "Double  strength"  infusion,  used  in  making 
agar-agar,  requires  equal  parts  of  the  meat  and  water. 

Infusion  Bouillon 

Beef  infusion 1000  c.c; 

Peptone  (Witte).  . -. 10  Gm.; 

Salt 5  Gm. 

Boil  until  dissolved;  bring  to  original  volume  with 
water;  adjust  reaction,  and  filter. 

Beef  Extract  Bouillon 

Liebig's  extract  of  beef 3  Gm. 

Peptone 10  Gm. 

Salt S  Gm. 

Tap- water 1000  c.c. 

Heat  until  all  ingredients  are  dissolved,  cool,  and  beat 
in  the  whites  of  two  eggs;  bring  slowly  to  the  boiling- 
point  again;  boil  briskly  for  five  minutes  and  filter.  It 
is  not  usually  necessary  to  adjust  the  reaction. 

Agar-agar 

Preparation  of  this  medium  usually  gives  the  student 
much  trouble.  There  should  be  no  difficulty  if  the 
directions  are  carefully  carried  out. 

Agar-agar,  powdered  or  in  shreds 15  Gm.; 

Tap- water 500  c.c. 

Boil  until  thoroughly  dissolved  and  add — 

Peptone 10  Gm.; 

Salt 5  Gm. 

When  these  have  dissolved,  replace  the  water  lost  in 


566  BACTERIOLOGIC   METHODS 

boiling,  cool  to  about  6o°C.,  and  add  500  c.c.  double- 
strength  beef  infusion.  Bring  slowly  to  the  boil,  adjust- 
ing the  reaction  meanwhile,  and  boil  for  at  least  five 
minutes.  Filter  while  hot  through  a  moderately  thick 
layer  of  absorbent  cotton  wet  with  hot  water  in  a  hot 
funnel.  A  piece  of  coarse  wire  gauze  should  be  placed 
in  the  funnel  underneath  the  cotton  to  give  a  larger 
filtering  surface.  This  medium  will  be  clear  enough  for 
ordinary  work.  If  an  especially  clear  agar  is  desired,  it 
can  be  filtered  through  paper  in  an  Arnold  sterilizer. 

Agar  can  also  be  made  by  boiling  15  Gm.  of  powdered 
agar  in  1000  c.c.  of  bouillon  until  dissolved,  replacing 
the  water  lost  in  boiling,  and  filtering  through  paper 
in  a  sterilizer.     It  can  be  cleared  with  egg  if  desired. 

Glycerin  Agar-agar 
To  1000  C.c.  melted  agar  add  60  to  70  c.c.  glycerin. 

Blood  Agar-agar 

The  simplest  way  to  prepare  this  is  to  smear  a  drop 
of  blood,  obtained  by  puncture  of  the  finger,  over  the 
surface  of  an  agar-slant,  and  to  incubate  over  night  to 
make  sure  of  sterility.  It  is  used  chiefly  for  growing  the 
influenza  bacillus.  It  may  be  noted  that  the  bacillus 
will  not  grow  well  on  blood  from  a  person  who  has 
recently  recovered  from  influenza. 

A  blood-agar  prepared  as  follows  is  more  satisfactory: 
Melt  5  c.c.  sterile  agar  in  a  culture-tube,  cool  to  45°C. 
in  a  water-bath,  add  i  c.c.  human  blood  (about  15 
drops),  and  mix  well.  Cool  in  an  inclined  position 
or  pour  into  Petri  plates.  Incubate  twelve  to  twenty- 
four  hours  to  make  sure  of  sterility. 


CULTURE-MEDIA  .  567 

Gelatin 

Dissolve  100  to  120  Gm.  "golden  seal"  gelatin  in  1000 
c.c.  nutrient  bouillon  with  as  little  heat  as  possible, 
adjust  the  reaction,  cool,  beat  in  the  whites  of  two  eggs, 
bring  slowly  to  the  boiling  point,  boil  for  a  few  minutes, 
and  filter  hot  through  filter-paper  wet  with  hot  water. 
Sterilize  in  an  Arnold  sterilizer  for  twenty  minutes  upon 
three  successive  days  and  cool  in  cold  water  after  each 
heating.  Keep  at  room  temperature  between  heatings. 
Sugar  Media 

Any  desired  sugar  may  be  added  to  bouillon,  agar,  or 
gelatin  in  proportion  of  10  Gm.  to  the  liter.  Dextrose 
is  most  frequently  required.  When  other  sugars  are 
added,  media  made  from  beef-extract  should  be  used, 
since  those  made  from  beef-infusion  contain  enough 
dextrose  to  cause  confusion. 

The  various  sugars  may  also  be  added  to  Dunham's 
peptone  medium  and  Hiss'  serum-water-litmus. 

Loffler's  Blood-serum 

Dextrose-bouillon  (i  per  cent.) i  part; 

Blood-serum 3  parts. 

Mix  and  tube.  Place  in  an  inspissator  at  the  proper 
slant  for  three  to  six  hours  at  80°  to  90°C.  When  firmly 
coagulated,  sterilize  in  the  usual  way.  A  large  "double- 
cooker"  makes  a  satisfactory  inspissator.  The  tubes 
are  placed  in  the  inner  compartment  upon  a  layer  of 
cotton  at  the  proper  slant,  a  lid  with  perforation  for  a 
thermometer  is  applied,  and  the  whole  is  weighted 
down  in  the  water  of  the  outer  compartment. 
.  Blood-serum  is  obtained  as  follows:  Beef  or  pig-blood 
is  collected  in  a  bucket  at   the  slaughter-house  and 


568  BACTERIOLOGIC    METHODS 

placed  in  an  ice-chest  until  coagulated.  The  clot  is  then 
gently  loosened  from  the  wall  of  the  vessel.  After 
about  twenty-four  hours  the  serum  will  have  separated 
nicely  and  can  be  siphoned  off.  It  is  then  stored  in 
bottles  with  a  little  chloroform  until  needed.  Red  cells, 
if  ^.bundant,  darken  the  medium,  but  do  no  harm. 

Solidified  blood-serum  is  probably  the  most  satisfac- 
tory medium  for  general  purposes.  Nearly  all  patho- 
genic organisms  grow  well  upon  it. 

Egg  Medium 

This  has  been  recommended  as  a  substitute  for 
solidified  blood-serum.  In  a  mortar  grind  thoroughly 
the  white  and  yolk  of  one  egg  with  10  to  15  c.c.  of  i  per 
cent,  dextrose  bouillon.  Place  in  tubes,  inspissate,  and 
sterilize  as  described  for  solidified  blood-serum. 

Litmus  Milk 

Fresh  milk  is  steamed  in  an  Arnold  sterilizer  for  half 
an  hour,  and  placed  in  the  ice-chest  over  night.  The 
milk  is  siphoned  oflf  from  beneath  the  cream,  and  suffi- 
cient aqueous  solution  of  litmus  or,  preferably,  azolit- 
min  is  added  to  give  a  blue-violet  color.  It  is  then 
tubed  and  sterilized. 

Potato 

Cylinders  about  3^  in.  diameter  are  cut  from 
potato  and  split  obliquely.  These  wedge-shaped  pieces 
are  soaked  over  night  in  running  water  and  placed,  broad 
ends  down,  in  large  tubes,  in  the  bottom  of  which  is 
placed  a  little  cotton  saturated  with  water.  They  are 
sterilized  for  somewhat  longer  periods  than  ordinary 
media. 


CULTURE-MEDIA  569 

Dunham's  Peptone  Solution 

Peptone 10  Gm.; 

Sail 5  Gm.; 

Water 1000  c.c. 

Dissolve  by  boiling;  filter,  tube,  and  sterilize. 

This  medium  is  used  to  determine  indol  production. 
To  a  twenty-four-  to  forty-eight-hour-old  culture  is 
added  5  to  10  drops  of  concentrated,  chemically  pure 
sulphuric  acid  and  i  c.c.  of  i :  10,000  solution  of  sodium 
nitrite.  Appearance  of  a  pink  color  shows  the  presence 
of  indol.  A  pink  color  before  the  nitrite  is  added  shows 
the  presence  of  both  indol  and  nitrites. 

Hiss'  Serum-water  Media 

Blood-serum i  part ; 

Water 3  parts. 

Warm  and  adjust  reaction  to  +0.2  to  +0.8.  Add 
litmus  or  azolitmin  solution  to  give  a  blue-violet  color. 
Finally,  add  i  per  cent,  of  inulin  or  any  desired  sugar. 
The  inulin  medium  is  very  useful  in  distinguishing  be- 
tween the  pneumococcus  and  streptococcus. 

Bile  Medium 

Ox-  or  pig-bile  is  obtained  at  the  slaughter-house, 
tubed,  and  sterilized.  This  is  used  especially  for  ^grow- 
ing typhoid  bacilli  from  the  blood  during  life.  The  fol- 
lowing is  probably  as  satisfactory  as  fresh  bile  and  is 
more  convenient: 

Inspissated  ox-bile  (Merck) 30.0  Gm.; 

Peptone 2.5  Gm. ; 

Water 250.0  c.c. 

Dissolve,  place  in  tubes,  and  sterilize. 


570  BACTERIOLOGIC    METHODS 

Reaction  of  Media. — The  chemical  reaction  of  the 
medium  exerts  a  marked  influence  upon  the  growth  of 
bacteria.  It  is  adjusted  after  all  ingredients  are  dis- 
solved by  adding  sufficient  caustic  soda  solution  to 
overcome  the  acidity  of  the  meat  and  other  substances 
used.  In  general,  the  most  favorable  reaction  lies  be- 
tween the  neutral  points  of  litmus  and  phenolphtha- 
lein,  representing  a  very  faint  alkalinity  to  litmus. 
In  routine  work  it  is  usually  suflacient  to  test  with 
litmus-paper.  When  greater  accuracy  is  demanded, 
the  following  method  should  be  used:  After  all  ingre- 
dients are  dissolved  and  the  loss  during  boiling  has 
been  replaced  with  water,  lo  c.c.  of  the  medium  are 
transferred  to  an  evaporating  dish,  diluted  with  40  c.c. 
of  water,  and  boiled  for  three  minutes  to  drive  off  carbon 
dioxid.  One  c.c.  of  0.5  per  cent,  alcoholic  solution  of 
phenolphthalein  is  then  added,  and  decinormal  sodium 
hydroxid  solution  is  run  in  from  a  buret  until  the  neutral 
point  is  reached,  indicated  by  the  appearance  of  a  per- 
manent pink  color.  The  number  of  cubic  centimeters 
of  decinormal  solution  required  to  bring  this  color 
indicates  the  number  of  cubic  centimeters  of  normal 
sodium  hydroxid  solution  which  will  be  required  to 
neutralize  100  c.c.  of  the  medium.  The  standard 
reaction  is  +1.5,  which  means  that  the  medium  must 
be  of  such  degree  of  acidity  that  1.5  c.c.  of  normal 
solution  would  be  required  to  neutralize  100  c.c.  This 
corresponds  to  faint  alkalinity  to  litmus.  Most 
pathogenic  bacteria  grow  better  with  a  reaction  of 
+  1.0  or  -|-o.8.  Example:  If  the  10  c.c.  which  were 
titrated  required  2  c.c.  of  decinormal  solution  to  bring 
the  pink  color,  the  reaction  is  +2 ;  and  0.5  c.c.  of  normal 


STAINING    METHODS  57 1 

sodium  hydroxid  must  be  added  to  each  lOO  c.c.  of  the 
medium  to  reduce  it  to  the  standard,  +i.5. 

Tubing  Culture-media. — The  finished  product  is 
stored  in  flasks  or  distributed  into  test-tubes.  This  is 
done  by  means  of  a  funnel  fitted  with  a  section  of  rubber 
tubing  with  a  glass  tip  and  a  pinch-cock.  Great  care 
must  be  exercised,  particularly  with  media  which  solid- 
ify, not  to  smear  any  of  them  upon  the  inside  of  the 
mouth  of  the  tube,  otherwise  the  cotton  stopper  will 
stick.  Tubes  are  generally  filled  to  a  depth  of  3  or  4  cm. 
For  stab-cultures  a  greater  depth  is  required. 

After  tubing,  all  culture-media  must  be  sterilized  as 
already  described.  Agar-tubes  are  cooled  in  a  slanting 
position  to  secure  the  proper  surface  for  inoculation. 

Storage  of  Cultture-media. — All  media  should  be 
stored  in  a  cool  place,  preferably  an  ice-chest.  Evapo- 
ration may  be  prevented  by  covering  the  tops  of  the 
tubes  with  tin-foil  or  with  the  rubber  caps  which  are  sold 
for  the  purpose;  or  the  cotton  stopper  may  be  pushed 
in  a  short  distance  and  a  cork  inserted. 

V.  STAINING  METHODS 

In  general,  bacteria  are  stained  to  determine  their 
morphology,  their  reaction  with  special  methods  {e.g., 
Gram's  method),  and  the  presence  or  absence  of  certain 
structures,  as  spores,  flagella,  and  capsules.  Staining 
methods  for  various  purposes  have  been  given  in 
previous  chapters  and  can  be  found  by  consulting  the 
Index.  The  formulae  of  the  staining  fluids  are  given 
in  the  Appendix. 

Method  of  Staining  for  Morphology. — The  following 
method  is  used  when  one  wishes  to  detect  the  presence 


572  BACTERIOLOGIC    METHOOS 

of  bacteria  or  to  study  their  morpholog> .  It  is  appli- 
cable both  to  films  from  cultures  and  to  smears  from 
pus  or  other  pathologic  material.  Any  simple  bacterial 
stain  may  be  used  but  Loeffler's  methylene  blue  or 
Pappenheim's  pyronin-methyl-green  will  generally  be 
found  more  satisfactory. 

1.  Make  a  thin  smear  upon  a  slide  or  cover-glass.  Heavy 
wax-pencil  marks  across  the  slide  will  limit  the  stain  to  any 
portion  desired. 

2.  Dry  in  the  air,  or  by  warming  high  above  the  flame, 
where  one  can  comfortably  hold  the  hand. 

3.  "Fix"  by  passing  the  preparation,  film  side  up,  rather 
slowly  through  the  flame  of  a  Bunsen  burner:  a  cover-glass 
three  times,  a  slide  about  twelve  times.  One  can  learn  to 
judge  the  proper  temperature  by  touching  the  glass  to  the 
back  of  the  hand  at  intervals.  If  the  film  takes  on  a 
brownish  discoloration,  most  marked  about  the  edges,  it 
has  been  scorched  and  is  worthless.  Smears  can  also  be 
fixed  by  flaming  with  alcohol,  as  described  for  blood-films 
(see  p,  307),  or  by  soaking  for  one  to  three  minutes  in  a 
saturated  solution  of  mercuric  chlorid  and  rinsing  well. 
The  last  avoids  all  possibility  of  spoiling  the  preparation  by 
scorching. 

4.  Apply  the  stain  for  the  necessary  length  of  time, 
generally  one-quarter  to  one  minute. 

5.  Wash  in  water. 

6.  Dry  by  waving  high  above  a  flame  or  by  blotting  with 
filter-paper. 

7.  Mount  by  pressing  the  cover,  film  side  down,  upon  a 
drop  of  Canada  balsam  or  immersion-oil  on  a  slide.  Slides 
may  be  examined  with  the  oil-immersion  lens  without  a 
cover-glass. 

Gram's  Method. — This  is  a  very  useful  aid  in  differ- 
entiating certain  bacteria  and  should  be  frequently  re- 


STAINING    METHODS  573 

sorted  to.  It  is  very  easy  and  should  not  be  the  bug- 
bear which  it  apparently  is  to  many  students.  It 
depends  upon  the  fact  that  when  treated  successively 
with  gentian-violet  and  iodin,  certain  bacteria  (owing 
to  formation  of  insoluble  compounds)  retain  the  stain 
when  subsequently  treated  with  alcohol,  whereas 
others  quickly  lose  it.  The  former  are  called  Gram- 
positive;  the  latter,  Gram-negative.  In  order  to  render 
Gram-negative  organisms  visible,  some  contrasting 
counterstain  is  commonly  applied,  but  this  is  not  a 
part  of  Gram's  method  proper. 

1.  Make  smears,  dry,  and  fix  by  heat  or  mercuric  chlorid. 

2.  Apply  carbol-gentian-violet,  anilin-gentian-violet  or 
formalin-gentian-violet  two  to  five  minutes.  The  last  is 
probably  least  satisfactory. 

3.  Wash  with  water. 

4.  Apply  Gram's  iodin  solution  one-half  to  two  minutes. 

5.  Wash  in  alcohol  until  the  purple  color  ceases  to  come 
off.  This  is  conveniently  done  in  a  watch-glass.  The  prepa- 
ration is  placed  in  the  alcohol,  face  downward,  and  one 
edge  is  raised  and  lowered  with  a  needle.  As  long  as  any 
color  is  coming  off,  purple  streaks  will  be  seen  diffusing  into 
the  alcohol  where  the  surface  of  the  fluid  meets  the  smear. 
If  forceps  be  used,  beware  of  stain  which  may  have  dried 
upon  them.  The  thinner  portions  of  smears  from  pus  should 
be  practically  colorless  at  this  stage.  Microscopically,  the 
nuclei  of  pus-corpuscles  should  retain  little  or  no  color.  // 
the  smears  resist  decolorization,  the  gentian-violet  andi  odin  solu- 
tion should  be  applied  for  a  shorter  time,  say,  one-half  minute 
each. 

6.  Apply  a  contrast  stain  for  one-half  to  one  minute. 
The  stains  commonly  used  for  this  purpose  are  an  aqueous 
or  alcoholic  solution  of  Bismarck  brown  and  a  weak  solution 


574  BACTERIOLOGIC    METHODS 

of  fuchsin.  A  i  per  cent,  aqueous  solution  of  safranin  is 
better.  In  the  writer's  experience,  Pappenheim's  pyro- 
nin-methyl-green  mixture  is  still  more  satisfactory;  it 
brings  out  Gram-negative  bacteria  sharply,  and  is  espe- 
cially desirable  for  intracellular  Gram-negative  organ- 
isms like  the  gonococcus  and  influenza  bacillus,  since  the 
^bacteria  are  bright  red  and  nuclei  of  cells  blue. 
7.  Wash  in  water,  dry,  and  mount  in  balsam. 

The  more  important  bacteria  react  to  this  staining 
method  as  follows: 

Gram  Staining  Gram  Decolorizing 

(Deep  purple)  (Colorless  unless  a  counterstain  is  used) 

Staphylococcus.  Gonococcus. 

Streptococcus.  Meningococcus. 

Pneumococcus.  Micrococcus  catarrhalis. 

Bacillus  diphtheriae.  Bacillus  of  influenza. 

Bacillus  tuberculosis.  Typhoid  bacillus. 

Bacillus  of  anthrax.  Bacillus  coli  communis. 

Bacillus  of  tetanus.  Spirillum  of  Asiatic  cholera. 

Bacillus  aerogenes  capsulatus.  Bacillus  pyocyaneus. 

Bacillus  of  Friedlander. 

Koch-Weeks  bacillus. 

Bacillus  of  Morax-Axenfeld. 

MSller's  Method  for  Spores. — Bodies  of  bacteria  are 
blue,  spores  are  red. 

1.  Make  thin  smears,  dry,  and  fix. 

2.  Wash  in  chloroform  for  two  minutes. 

3.  Wash  in  water. 

4.  Apply  5  per  cent,  solution  of  chromic  acid  one-half  to 
two  minutes. 

5.  Wash  in  water. 

6.  Apply  carbolfuchsin  and  heat  to  boiling. 

7.  Decolorize  in  5  per  cent,  solution  of  sulphuric  acid. 

8.  Wash  in  water. 


STAINING    METHODS  575 

9.  Apply  I  per  cent,  aqueous  solution  of  methylene-blue 
one-half  minute. 

10.  Wash  in  water,  dry,  and  mount. 

Himtoon's  Method  for  Spores. — This  method  is 
simple  and  appears  to  be  very  reliable.  Spores  are 
deep  red,  bodies  of  bacteria  are  blue. 

1.  Make  a  rather  thick  smear,  dry,  and  fix  in  the  usual 
way. 

2.  Apply  as  much  of  the  stain  as  will  remain  on  the 
cover-glass,  and  steam  over  a  flame  for  one  minute,  replacing 
the  stain  lost  by  evaporation. 

3.  Wash  in  water.     The  film  is  bright  red. 

4.  Dip  the  preparation  a  few  times  into  a  weak  solution 
of  sodium  carbonate  (7  or  8  drops  of  saturated  solution  in  a 
glass  of  water).  Too  long  application  of  the  carbonate  will 
cause  the  spores  to  be  blue. 

5.  The  instant  the  film  turns  blue,  rinse  well  in  water. 

6.  Dry,  mount,  and  examine. 
Preparation  of  Stain. — 

(i)  Acid  fuchsin  (Griibler) 4  Gm.; 

Aqueous  solution  acetic  acid  (2  per  cent.).   50  c.c. 

(2)  Methylene-blue  (Griibler) 2  Gm.; 

Aqueous  solution  acetic  acid  (2  per  cent.).  50  c.c. 

Mix  the  two  solutions,  let  stand  for  fifteen  minutes,  and 
filter  off  the  voluminous  precipitate  through  moistened 
filter-paper.  The  filtrate  is  the  staining  fluid.  It  keeps 
several  weeks,  but  requires  filtration  when  a  precipitate 
forms. 

Lofller's  Method  for  Flagella. — The  methods  for 
fiagella  are  applicable  only  to  cultures.  Enough  of  the 
growth  from  an  agar-culture  (which  should  not  be  more 
than  eighteen  to  twenty-four  hours  old)  to  produce  faint 


576  BACTERIOLOGIC   METHODS 

cloudiness  is  added  to  distilled  water.  A  small  drop  of 
this  is  placed  on  a  cover-glass,  spread  by  tilting,  and 
dried  quickly.  The  covers  must  be  absolutely  free 
from  grease.  To  insure  this,  they  may  be  warmed  in 
concentrated  sulphuric  acid,  washed  in  water,  and 
kept  in  a  mixture  of  alcohol  and  strong  ammonia. 
When  used  they  are  dried  upon  a  fat-free  cloth. 
Covers  may  be  dried  without  touching  them  with  the 
fingers  by  rubbing  between  two  blocks  of  wood  covered 
with  several  layers  of  lint-free  cloth. 

1.  Fix  by  heating  the  cover  over  a  flame  while  holding 
in  the  fingers. 

2.  Cover  with  freshly  filtered  mordant  and  gently  warm 
for  about  a  minute. 

The  mordant  consists  of: 

Aqueous  solution  of  tannic  acid  (20  per  cent.) 10  c.c; 

Saturated  solution  ferrous  sulphate,  cold 5  c.c; 

Saturated  aqueous  or  alcoholic  solution  gentian- 
violet 1  c.c. 

3.  Wash  in  water. 

4.  Apply  freshly  filtered  anilin-gentian-violet,  warming 
gently  for  one-half  to  one  minute. 

5.  Wash  in  water,  dry,  and  mount  in  balsam. 

VI.  METHODS  OF  STUDYING  BACTERIA 

The  purpose  of  bacteriologic  examinations  is  to  de- 
termine whether  bacteria  are  present  or  not,  and,  if 
present,  their  species  and  comparative  numbers.  In 
general,  this  is  accomplished  by:  (i)  direct  micro- 
scopic examination;  (2)  cultural  methods;  (3)  animal 
inoculation. 


METHODS    OF    STUDYING   BACTERIA  577 

1.  Direct  Microscopic  Examination. — Every  bac- 
teriologic  examination  should  begin  with  a  microscopic 
study  of  smears  from  the  pathologic  ma,terial,  stained 
with  a  general  stain,  by  Gram's  method,  and  often  by 
the  method  for  the  tubercle  bacillus.  This  yields  a 
great  deal  of  information  to  the  experienced  worker, 
and  in  many  cases  is  all  that  is  necessary  for  the  pur- 
pose in  view.  It  will  at  least  give  a  general  idea  of  what 
is  to  be  expected,  and  may  determine  future  procedure. 

2.  Cultural  Methods. — i.  Collection  of  Material. — 
Material  for  examinations  must  be  collected  under  abso- 
lutely aseptic  conditions.  It  may  be  obtained  with  a 
platinum  wire — which  has  been  heated  to  redness  just 
previously  and  allowed  to  cool — or  with  a  swab  of  sterile 
cotton  on  a  stiff  wire  or  wooden  applicator.  Such 
swabs  may  be  placed  in  cotton-stoppered  test-tubes, 
sterilized,  and  kept  on  hand  ready  for  use.  Fluids 
which  contain  very  few  bacteria,  and  hence  require 
that  a  considerable  quantity  be  used,  may  be  collected 
in  a  sterile  hypodermic  syringe  or  one  of  the  pipets 
described  on  page  561.  The  method  of  obtaining 
blood  for  cultures  is  given  on  pp.  346,  347. 

2.  Inoculating  Media. — The  material  is  thoroughly 
distributed  over  the  surface  of  some  solid  medium, 
solidified  blood-serum  being  probably  the  best  for 
routine  work.  When  previous  examination  of  smears 
has  shown  that  many  bacteria  are  to  be  expected,  a 
second  tube  should  be  inoculated  from  the  first,  and  a 
third  from  the  second,  so  as  to  obtain  isolated  colonies 
in  at  least  one  of  the  tubes.  The  platinum  wire  must 
be  heated  to  redness  before  and  after  each  inoculation. 
When  only  a  few  organisms  of  a  single  species  are  ex- 
37 


578  BACTERIOLOGIC   METHODS 

pected,  as  is  the  case  in  blood-cultures  (see  p.  347),  a 
considerable  quantity  of  the  material  is  mixed  with  a 
fluid  medium. 

3.  Incubation. — Cultures  are  pJaced  in  an  incubator 
which  maintains  a  uniform  temperature,  usually  of 
37.5°C.,  for  eighteen  to  twenty-four  hours,  and  the 
growth,  if  any,  is  studied  as  described  later.  Gelatin 
will  melt  with  this  degree  of  heat,  and  must  be  in- 
cubated at  about  room-temperature. 

4.  Study  of  Cultures. — When  the  original  culture 
contains  more  than  one  species,  they  must  be  separated, 
or  obtained  in  "pure  culture,"  before  they  can  be 
studied  satisfactorily.  To  accomplish  this  it  is  neces- 
sary to  so  distribute  them  on  solid  media  that  they 
form  separate  colonies,  and  to  inoculate  fresh  tubes 
from  the  individual  colonies.  In  routine  work  the 
organisms  can  be  sufficiently  distributed  by  drawing  the 
contaminated  wire  over  the  surface  of  the  medium  in  a 
series  of  streaks.  If  a  sufficient  number  of  streaks  be 
made,  some  of  them  are  sure  to  show  isolated  colonies. 
Another  method  of  obtaining  isolated  colonies  is  to 
inoculate  the  water  of  condensation  of  a  series  of  tubes, 
the  first  from  the  second,  the  second  from  the  third, 
etc.,  and,  by  tilting,  to  flow  the  water  once  over  the 
surface  of  the  medium.  One  or  more  of  these  tubes 
will  be  almost  sure  to  show  nicely  separated  colonies. 

In  order  to  determine  the  species  to  which  an  organ- 
ism belongs  it  is  necessary  to  consider  some  or  all  of  the 
following  points: 

1.  Naked-eye  and  microscopic  appearance  of  the  col- 
onies on  various  media. 

2.  Comparative  luxuriance  of  growth  upon  various 


METHODS    OF    STUDYING   BACTERIA  579 

media.  The  influenza  bacillus,  for  example,  can  be 
grown  upon  media  containing  hemoglobin,  but  not  on 
the  ordinary  media. 

3.  Morphology,  special  staining  reactions,  and  the 
presence  or  absence  of  spores,  flagella,  and  capsules. 
Staining  methods  for  these  purposes  have  been  given. 

4.  Motility.  This  is  determined  by  observing  the 
living  organism  with  an  oil-immersion  lens  in  a  hanging- 
drop  preparation,  made  as  follows:  A  small  drop  of  a 
bouillon  culture  or  of  water  of  condensation  from  an 
agar  or  blood-serum  tube  is  placed  upon  the  center  of 
a  cover-glass;  and  over  this  is  pressed  the  concavity  of 
a  ''hollow-ground  slide"  previously  ringed  with  vaselin. 
The  slide  is  then  turned  over  so  as  to  bring  the  cover- 
glass  on  top.  In  focusing,  the  edge  of  the  drop  should 
be  brought  into  the  field.  Great  care  must  be  exer- 
cised not  to  break  the  cover  by  pushing  the  objective 
against  it. 

It  is  not  always  easy  to  determine  whether  an  organ- 
ism is  or  is  not  motile,  since  the  motion  of  currents  and 
the  Brownian  motion  which  aflfects  all  particles  in  sus- 
pension are  sometimes  very  deceptive. 

5.  Production  of  chemical  changes  in  the  media. 
Among  these  are  coagulation  of  milk ;  production  of  acid 
in  milk  and  various  sugar  media  to  which  litmus  has 
been  added  to  detect  the  change;  production  of  gas  in 
sugar  media,  the  bacteria  being  grown  in  fermentation 
tubes  similar  to  those  used  for  sugar  tests  in  urine;  and 
production  of  indol. 

6.  Ability  to  grow  without  free  oxygen. 

5.  Anaerobic  Methods.— Some  bacteria,  the  "ob- 
ligate anaerobes,"  will  not  grow  unless  free  oxygen  is  ex- 


580  BACTERIOLOGIC   METHODS 

eluded.  This  may  be  accomplished  in  various  ways. 
Perhaps  the  most  convenient  is  the  following  method 
of  J.  H.  Wright.  After  the  culture  medium  in  the 
test-tul*e  has  been  inoculated,  push  the  cotton  stopper 
in  until  its  top  is  about  1.5  cm.  below  the  mouth  of  the 
tube.  Fill  in  the  space  above  the  stopper  with  dry 
pyrogallic  acid  and  pour  on  it  just  enough  strong 
solution  of  sodium  hydroxid  to  dissolve  it.  Finally, 
insert  a  rubber  cork  and  seal  with  paraffin. 

7.  Effects  produced  when  inoculated  into  animals. 

3.  Animal  Inoculation.^ — In  clinical  work  this  is 
resorted  to  chiefly  to  detect  the  tubercle  bacillus.  The 
method  is  described  on  page  534. 

For  the  study  of  bacteria  in  cultures,  a  small  amount 
of  a  pure  culture  is  injected  subcutaneously  or  into  the 
peritoneal  cavity.  The  animals  most  used  are  guinea- 
pigs,  rabbits,  and  mice.  For  intravenous  injection,  the 
rabbit  is  used  because  of  the  easily  accessible  marginal 
vein  of  the  ear. 

VII.  CHARACTERISTICS  OF  SPECIAL  BACTERIA 

Owing  to  the  great  number  of  bacterial  species,  most 
of  which  have  not  been  adequately  studied,  positive 
identification  of  an  unknown  organism  is  often  a  very 
difficult  problem.  Fortunately,  however,  only  a  few 
are  commonly  encountered  in  routine  work,  and  identi- 
fication of  these  with  comparative  certainty  presents  no 
great  difficulty.  Their  more  distinctive  characteristics 
are  outlined  in  this  section. 

1.  Staphylococcus  pyogenes  aureus. — The  mor- 
phology and  staining  reactions  (described  on  p.  516)  and 
the  appearance  of  the  colonies  are  sufficient  for  diag- 


CHARACTERISTICS    OF    SPECIAL   BACTERIA  58 1 

nosis.  Colonies  on  solidified  blood-serum  and  agar 
are  rounded,  slightly  elevated,  smooth  and  shining, 
and  vary  in  color  from  light  yellow  to  deep  orange. 
Young  colonies  are  sometimes  white. 

2.  Staphylococcus  pyogenes  albus. — This  is  simi- 
lar to  the  above,  but  colonies  are  white.  It  is  generally 
less  virulent. 

3.  Staphylococcus  pyogenes  citreus. — The  colo- 
nies are  lemon  yellow;  otherwise  it  resembles  the  white 
staphylococcus. 

4.  Streptococcus  pyogenes. — The  morphology  and 
staining  reactions  have  been  described  (see  p.  516). 
The  chains  are  best  seen  in  the  water  of  condensation 
and  in  bouillon  cultures.  The  cocci  are  not  motile. 
Colonies  on  blood-serum  are  minute,  round,  grayish, 
and  translucent.  Litmus  milk  is  usually  acidified  and 
coagulated,  although  slowly.  The  streptococcus  rarely 
produces  acid  in  Hiss'  serum-water-litmus-inulin 
medium  (see  p.  569). 

Some  strains  of  streptococcus  are  capable  of  hemo- 
lyzing  red  blood  corpuscles,  and  this  property  is 
utilized  in  classification.  Hemolysis  is  manifested  by 
the  appearance  of  a  wide  clear  zone  around  the  colonies 
when  grown  upon  blood-agar.  Many  non-hemolyzing 
streptococci  produce  a  narrow  green  zone  upon  this 
medium,  and  these  are  grouped  under  the  name  Strepto- 
coccus viridans.  Such  streptococci  are  less  actively 
virulent  than  the  hemolyzing  type,  being  most  fre- 
quently associated  with  chronic  inflammations. 

5.  Pneumococcus. — The  only  organism  with  which 
this  is  likely  to  be  confused  is  the  streptococcus.  The 
distinction  is  often  extremely  difficult. 


582  BACTERIOLOGIC    METHODS 

Detection  of  the  pneumococcus  in  fresh  material  has 
been  described  (see  p.  85).  In  cultures  it  frequently 
forms  long  chains.  Capsules  are  not  present  in  cultures 
except  upon  special  media.  They  show  best  upon  a 
serum-medium  like  that  described  for  the  gonococcus, 
but  can  frequently  be  seen  in  milk.  Colonies  on 
blood-serum  resemble  those  of  the  streptococcus. 
Colonies  on  blood-agar  show  a  green  zone  like  those 
of  Streptococcus  viridans.  The  pneumococcus  usually 
promptly  acidifies  and  coagulates  milk  and  acidifies 
and  coagulates  Hiss'  serum-water  with  inulin.  The 
latter  property  is  very  helpful  in  diagnosis. 

6.  Micrococcus  catarrhalis  grows  readily  at  room 
temperature  and  on  ordinary  media  where  it  forms 
large,  white,  dry  colonies  with  irregular  edges  and 
elevated  centers.  This  readily  distinguishes  it  from 
the  gonococcus  and  meningococcus,  which  it  closely 
resembles  in  morphology  and  staining  reactions. 

7.  Gonococcus. — Its  morphology  and  staining  pecu- 
liarities are  given  on  page  518.  These  usually  suffice 
for  its  identification,  cultural  methods  being  rarely 
undertaken.  In  cultures  the  chief  diagnostic  point 
is  its  failure  to  grow  on  ordinary  media.  To  grow 
it,  the  most  convenient  medium  is  made  by  adding 
ascitic  or  hydrocele  fluid  (which  has  been  obtained 
under  aseptic  conditions)  to  melted  agar  in  proportion 
of  I  part  of  serum  to  3  parts  of  agar.  The  agar  in 
tubes  is  melted  and  cooled  to  about  45°C.;  the  serum 
is  added  with  a  pipet  and  mixed  by  shaking;  and  the 
tubes  are  again  cooled  in  a  slanting  position.  Colonies 
upon  this  medium  are  minute,  gra5Hlsh,  and  translucent. 


CHARACTERISTICS    OF    SPECIAL   BACTERIA  583 

8.  Diplococcus  intracellularis  meningitidis. — ^It 

grows  poorly  or  not  at  all  on  plain  agar.  On  Loffler's 
blood-serum,  upon  which  it  grows  fairly  well,  colonies 
are  round,  colorless  or  hazy,  flat,  shining,  and  viscid- 
looking.     It  quickly  dies  out. 

9.  Diphtheria  Bacillus. — The  diagnosis  is  usually 
made  from  a  study  of  stained  smears  from  cultures 
upon  blood-serum,  giown  for  twelve  to  eighteen  hours. 
Its  morphology  and  staining  peculiarities  are  character- 
istic when  grown  on  this  medium  (see  p.  538).  The 
bacilli  are  non-motile  and  Gram-positive.  The  colonies 
are  round,  elevated,  smooth,  and  grayish. 

10.  Typhoid  and  Colon  Bacilli. — These  are 
medium-sized,  motile,  Gram-negative,  non-spore-bear- 
ing bacilli.  Upon  blood-serum  they  form  rounded, 
grayish,  slightly  elevated,  viscid  looking  colonies,  those 
of  the  colon  bacillus  being  somewhat  the  larger.  They 
do  not  liquefy  gelatin;  They  represent  the  extremes 
of  a  large  group  with  many  intermediate  types.  They 
are  distinguished  as  follows: 

Typhoid  Bacillus  Colon  Bacillus 

Actively  motile.  Much  less  active. 

Growth  on  potato  usually  invisible.  Growth  distinctly  visible  as  dirty 

gray  or  brownish  slimy  layer. 

No  gas  produced  in  glucose  media.  Produces  gas. 

Growth  in  litmus  milk  produces  no  Litmus  milk  pink  and  coagulated. 

change. 

Produces    no    indol    in    Dunham's  Produces   indol.     (For  test,  see 

peptone  medium.  p.  569.) 

Agglutinates  with  serum  from  ty-  Does  not  agglutinate  with  typhoid 

phoid-fever    patient.     (Recently        serum. 

isolated  bacilli  do  not  agglutinate 

well.) 


584  BACTERIOLOGIC    METHODS 

11.  Bacillus  of  Influenza. — Diagnosis  will  usually 
rest  upon  the  morphology  and  staining  peculiarities,  de- 
scribed on  page  89,  and  upon  the  fact  that  the  bacillus 
will  not  grow  on  ordinary  media,  but  does  grow  upon 
hemoglobin-containing  media.  It  can  be  grown  upon 
agar-slants  which  have  been  smeared  with  a  drop  of 
blood  from  a  puncture  in  the  finger.  Before  inocula- 
tion these  slants  should  be  incubated  to  make  sure  of 
sterility.  The  colonies  are  difficult  to  see  without  a 
hand  lens.  They  are  very  minute,  discrete,  and  trans- 
parent, resembling  small  drops  of  dew. 

12.  Bacillus  of  Tuberculosis. —  The  methods  of 
identifying  this  important  organism  have  been  given  (see 
pp.  76,  235).  Cultivation  is  not  resorted  to  in  routine 
clinical  work.  It  grows  very  slowly  and  only  on  certain 
media.     It  is  Gram-positive  and  non-motile. 


CHAPTER  IX 

PREPARATION  AND  USE  OF  VACCINES 

Bacterial  vaccines,  sometimes  called  "bacterins," 
which  within  recent  years  have  come  to  play  an  im- 
portant role  in  therapeutics,  are  suspensions  of  defi- 
nite numbers  of  dead  bacteria  in  normal  salt  solution. 
While  in  many  cases,  notably  in  gonorrhea  and  tuber- 
culosis, ready  prepared  or  ''stock"  vaccines  are  satis- 
factory, it  is  usually  desirable  and  often  imperative  for 
best  results  to  use  vaccines  which  are  especially  pre- 
pared for  each  patient  from  bacteria  which  have  been 
freshly  isolated  from  his  own  lesion.  These  latter  are 
called  "autogenous  vaccines."  Only  through  them  can 
one  be  certain  of  getting  the  exact  strain  of  bacterium 
which  is  producing  the  disease. 

I.    PREPARATION  OF  VACCINE 

1.  Preparation  of  Materials. — A  number  of  2- 
ounce  wide-mouthed  bottles  are  cleaned  and  sterilized. 
Each  is  charged  with  50  c.c.  freshly  filtered  normal  salt 
solution  (0.85  per  cent,  sodium  chlorid  in  distilled  water) , 
and  is  capped  with  a  new  rubber  nursing-nipple,  with- 
out holes,  inverted  as  shown  in  Fig.  2  26.  A  small  section 
of  hollow  wire  or  a  hypodermic  needle  is  thrust  through 
the  cap  near  the  edge  to  serve  as  ah  air  vent,  and  the 
bottle  and  contents  are  sterilized  in  an  autoclave.  If 
an  autoclave  is  not  at  hand,  successive  steamings  in  an 


586 


PREPARATION    AND    USE    OF   VACCINES 


Arnold  sterilizer  will  answer,  provided  it  is  not  opened 
between  steamings.  After  sterilization,  the  pieces  of 
wire  are  pulled  out  and  the  holes  sealed  with  collodion. 
Most  workers  use  a  smaller  bottle  with  less  salt  solu- 
tion and  with  a  cotton  stopper;  and,  after  the  solution 
has  been  sterilized,  apply  a  specially  made  rubber 
"vaccine-bottle  cap."     Instead  of  the  cotton  stopper,  a 


Fig.  226. — -Vaccine  bottles:  A,  Cap  ready  to  be  applied;  B,  ready  for 
sterilization;   C,  finished  vaccine. 

sheet  of  paper  which  is  placed  over  the  top,  folded 
closely  about  the  neck  of  the  bottle,  and  held  in  place 
with  a  rubber  band  may  advantageously  be  used  as  a  tem- 
porary cap.  The  first  method  calls  for  an  unnecessarily 
large  quantity  of  fluid  (which  is  no  real  objection),  but 
has  certain  slight  advantages:  the  nursing  nipples  are 
easily  obtained  at  any  pharmacy;  the  rubber  is  not  put 


PREPARATION    OF   VACCINE  587 

upon  the  stretch  as  is  the  case  with  some  caps,  and  is, 
therefore,  self-sealing;  no  cotton-Hnt  falls  into  the  salt 
solution  before  the  cap  is  applied;  and  the  cap  offers  a 
concavity  which  may  be  filled  with  80  per  cent,  alcohol 
for  sterilizing  before  the  needle  is  plunged  through, 

A  number  of  test-tubes,  each  charged  with  10  c.c.  of 
normal  salt  solution  and  plugged  with  cotton,  are  also 
prepared  and  sterilized. 

2.  Obtaining  the  Bacteria. — A  culture  on  some 
solid  medium  is  made  from  the  patient's  lesion,  and  a 
pure  culture  is  obtained  in  the  usual  way.  This  pre- 
liminary work  should  be  carried  through  as  quickly  as 
possible  in  order  that  the  bacteria  may  not  lose  viru- 
lence by  long  growth  upon  artificial  media.  Tf  for  any 
reason  there  is  much  delay,  it  i.s  best  to  begin  over 
again,  the  experience  gained  in  the  first  trial  enabling 
one  to  carry  the  second  through  more  rapidly.  When 
a  pure  culture  is  obtained,  a  number  of  tubes  of  blood- 
serum  or  agar — 10  or  12  in  the  case  of  streptococcus 
or  pneumococcus,  4  or.  5  in  the  case  of  most  other 
organisms — are  planted  and  incubated  over  night  or 
until  a  good  growth  is  obtained. 

3.  Making  the  Suspension.^ — A  few  cubic  centi- 
meters of  the  salt  solution  from  one  of  the  lo-c.c.  salt- 
tubes  is  transferred  by  means  of  a  sterile  pipet  to  each  of 
the  culture-tubes,  and  the  growth  thoroughly  rubbed  up 
with  a  stiff  platinum  wire  or  a  glass  rod  whose  tip  is  bent 
at  right  angles.  The  suspension  from  the  different 
tubes,  usually  amounting  to  about  10  c.c,  is  then  col- 
lected in  one  large  tube  (size  about  150  X  19  mm.) ;  and 
the  upper  part  of  the  tube  is  drawn  out  in  the  flame  of  a 
blast  lamp  or  Bunsen  burner,  as  indicated  in  Fig.  227,  J5, 


588  PREPARATION    AND   USE    OF  VACCINES 

a  short  section  of  glass  tubing  being  fused  to  the  rim  of 
the  tube  to  serve  as  a  handle.  It  is  then  stood  aside, 
and  when  cool  the  narrow  portion  is  sealed  ofT. 

The  resulting  hermetically  sealed  capsule  is  next 
thoroughly  shaken  for  ten  to  twenty  minutes  to  break 
up  all  clumps  of  bacteria.     Some  small  pieces  of  glass 


Fig.  227. — Process  of  making  hermetically  sealed  capsules  containinjf 

liquid. 

or  a  little  clean  sterile  sand  may  be  introduced  to  assist 
in  this,  but  with  many  organisms  it  is  not  necessary. 

4.  Sterilization. — The  capsule  is  placed  in  a  water- 
bath  at  6o°C.  for  forty-five  minutes.  This  can  be  done 
in  an  ordinary  rice-cooker,  with  double  lid,  through 
which  a  thermometer  is  inserted.  When  both  compart- 
ments are  filled  with  water  it  is  an  easy  matter  to  main- 
tain a  uniform  temperature  by  occasional  application  of  a 
small  flame.     The  time  and  temperature  are  important: 


PREPARATION    OF  VACCINE 


589 


too  little  heat  will  fail  to  kill  the 
bacteria,  and  too  much  will  destroy 
the  efficiency  of  the  vaccine. 

When  sterilization  is  complete  the 
capsule  is  opened,  a  few  drops  are 
planted  on  agar  or  blood-serum,  and 
the  capsule  is  again  sealed. 

5.  Counting. — When  incubation 
of  the  planted  tube  has  shown  the 
suspension  to  be  sterile  it  is  ready  for 
counting.  Of  the  two  methods  given 
here  the  latter  is  the  more  accurate. 

Wright's  Method. — There  must  be 
ready  a  number  of  clean  slides;  a  few 
drops  of  normal  salt  solution  on  a  slide 
or  in  a  watch-glass;  a  blood-lancet, 
which  can  be  improvised  from  a  spicule 
of  glass  or  a  pen;  and  two  capillary 
pipets  with  squarely  broken  off  tips  and 
wax-pencil  marks  about  2  cm.  from 
the  tip  (Fig.  228).  These  are  easily 
made  by  drawing  out  a  piece  of  glass 
tubing,  as  described  on  page  561. 

It  is  necessary  to  work  quickly.  After 
thorough  shaking,  the  capsule  is  opened 
and  a  few  drops  forced  out  upon  a  slide. 
Any  remaining  clumps  of  bacteria  are 
broken  up  with  one  of  the  pipets  by 
holding  it  against  and  at  right  angles 
to  the  slide,  and  alternately  sucking  the 
fluid  in  and  forcing  it  out.  The  pipet 
is  most  easily  controlled  if  held  in  the 
whole  hand  with  the  rubber  bulb  between 


3  2 


O  *< 

2  ^ 
2. 

2  3 


CL  3 


590     PREPARATION  AND  USE  OF  VACCINES 

the  thumb  and  the  side  of  the  index-finger.  A  finger  is  then 
pricked  until  a  drop  of  blood  appears;  and  into  the  second 
pipet  are  quickly  drawn  successively:  i  or  2  volumes 
normal  salt  solution  (or,  better,  a  i  per  cent,  solution  of 
sodium  citrate,  which  prevents  coagulation) ;  a  small  bubble 
of  air;  i  volume  of  blood;  a  small  bubble  of  air;  and,  finally, 
I  volume  of  bacterial  suspension.  (A  "  volume  "  is  measured 
by  the  distance  from  the  tip  of  the  pipet  to  the  wax- 
pencil  mark.)  The  contents  of  the  pipet  are  then  forced 
out  upon  a  slide  and  thoroughly  mixed  by  sucking  in  and 
out,  care  being  taken  to  avoid  bubbles;  after  which  the 
fluid  is  distributed  to  a  number  of  sides  and  spread  as  in 
making  blood-smears. 

The  films  are  stained  with  Wright's  blood-stain  or, 
better,  by  a  few  minutes'  application  of  carbol-thionin, 
after  fiixing  for  a  minute  in  saturated  mercuric  chlorid 
solution.  With  an  oil-immersion  lens  both  the  red  cells 
and  the  bacteria  in  a  number  of  microscopic  fields  are 
counted.  The  exact  number  is  not  important;  for  con- 
venience 500  red  cells  may  be  counted.  From  the  ratio 
between  the  number  of  bacteria  and  of  red  cells,  it  is 
easy  to  calculate  the  number  of  bacteria  in  i  c.c.  of  the 
suspension,  it  being  known  that  there  are  5000  million 
red  corpuscles  in  a  cubic  centimeter  of  normal  human  blood. 
If  there  were  twice  as  many  bacteria  as  red  corpuscles  in 
the  fields  counted,  the  suspension  would  contain  10,000 
million  bacteria  per  cubic  centimeter. 

Hemacytometer  Method. — This  is  carried  out  in  the  same 
manner  as  a  blood  count,  using  any  convenient  dilution, 
usually  I  :  200.  A  weak  carbol-f uchsin  or  gentian-violet 
solution,  freshly  filtered,  may  be  used  as  diluting  fluid,  but 
the  following  solution,  recommended  by  Callison  is  better: 

Hydrochloric  acid 2  c.c; 

Mercuric  chlorid  (0.2  per  cent,  solution) ....    100  c.c; 
Acid  fuchsin  (i  per  cent,  aqueous  solution),  to  color. 
The  color  should  be  just  deep  enough  not  to  obscure  the  ruled  lines. 


METHOD    OF    USE  59I 

A  very  thin  cover-glass  must  be  used;  and,  after  filling, 
the  counting-chamber  must  be  set  aside  for  an  hour  or 
more  to  allow  the  bacteria  to  settle.  Mallory  and  Wright 
advise  the  use  of  the  shallow  Helber  chamber  manu- 
factured by  Zeiss  for  counting  blood-plates,  but  many 
2-mm.  oil-immersion  objectives  have  sufficient  working  dis- 
tance to  allow  the  use  of  the  regular  counting-chamber, 
provided  a  very  thin  cover  is  used.  The  heavy  cover  with 
central  excavation  is  recommended. 

6.  Diluting. — The  amount  of  the  suspension  which, 
when  diluted  to  50  c.c,  will  give  the  strength  desired  for 
the  finished  vaccine  having  been  determined,  this 
amount  of  salt  solution  is  withdrawn  with  a  hypodermic 
syringe  from  one  of  the  bottles  already  prepared,  and  is 
replaced  with  an  equal  amount  of  suspension.  One- 
tenth  cubic  centimeter  of  trikresol  or  lysol  is  finally 
added  and  the  vaccine  is  ready  for  use.  To  prevent 
possible  leakage  about  the  cap,  the  neck  of  the  bottle 
is  dipped  in  melted  paraffin.  The  usual  strengths  are: 
staphylococcus,  1000  million  in  i  c.c;  most  other 
bacteria,  100  million  in  i  c.c. 

II.  METHOD  OF  USE 

Vaccines  are  administered  subcutaneously,  usually  in 
the  arm  or  abdominal  wall  or  between  the  shoulder- 
blades.  The  technic  is  the  same  as  for  an  ordinary 
hypodermic  injection.  The  syringe  is  usually  sterilized 
by  boiling.  The  site  of  the  injection  may  be  mopped  with 
alcohol  or  may  be  touched  with  a  pledget  of  cotton 
saturated  with  tincture  of  iodin  or  liquor  cresolis  com- 
positus.  The  rubber  cap  of  the  container  is  sterilized 
by  filling  the  concavity  with  alcohol  for  some  minutes, 


592  PREPARATION   AND   USE    OF  VACCINES 

usually  while  the  syringe  is  being  sterilized,  or  simply 
placing  a  drop  of  liquor  cresolis  compositus  upon  it. 
The  bottle  is  then  inverted  and  well  shaken,  when  the 
needle  is  plunged  through  the  rubber  and  the  desired 
quantity  withdrawn.  The  hole  seals  itself.  A  satis- 
factory syringe  is  the  comparatively  inexpensive 
Luer  I  c.c.  "Tuberculin"  syringe  graduated  in  hundredths 
of  a  cubic  centimeter. 

III.  DOSAGE 

Owing  to  variations,  both  in  virulence  of  organisms 
and  susceptibility  of  patients,  no  definite  dosage  can  be 
assigned.  Each  case  is  a  separate  problem.  Wright's 
original  proposal  was  to  regulate  the  size  and  frequency 
of  dose  by  its  effect  upon  the  opsonic  index,  but  this  is 
beyond  the  reach  of  the  practitioner.  The  more  widely 
used  "clinical  method"  consists  in  beginning  with  a 
very  small  dose  and  cautiously  increasing  until  the 
patient  shows  either  improvement  or  some  sign  of  a 
"reaction,"  indicated  by  headache,  malaise,  fever,  ex- 
acerbation of  local  disease,  or  inflammatory  reaction  at 
the  site  of  injection.  The  reaction  indicates  that  the 
dose  has  been  too  large.  The  beginning  dose  of  staphy- 
lococcus is  about  50  million;  the  maximum,  1000  million 
or  more.  Of  most  other  organisms  the  beginning  dose 
is  5  million  to  10  million;  maximum,  about  100  million. 
Ordinarily,  injections  are  given  once  or  twice  a  week ; 
very  small  doses  may  be  given  every  other  day. 

IV.  THERAPEUTIC  INDICATIONS 

The  therapeutic  effect  of  vaccines  depends  upon  their 
power  to  produce  active  immunity:  they  stimulate  the 


THERAPEUTIC   INDICATIONS  593 

production  of  opsonins  and  other  antibacterial  sub- 
stances which  enable  the  body  to  combat  the  infecting 
bacteria.  Their  especial  field  is  the  treatment  of  sub- 
acute and  chronic  localized  infections,  in  some  of  which 
they  offer  the  most  effective  means  of  treatment  at  our 
command.  In  most  chronic  infections  the  circulation  of 
blood  and  lymph  through  the  diseased  area  is  very 
sluggish,  so  that  the  antibodies,  when  formed,  cannot 
readily  reach  the  seat  of  disease.  Ordinary  measures 
which  favor  circulation  in  the  diseased  part  should, 
therefore,  accompany  the  vaccine  treatment.  Among 
these  may  be  mentioned  incisions  and  drainage  of  ab- 
scesses, dry  cupping,  application  of  heat.  Bier's  hyper- 
emia, etc.,  but  such  measures  should  not  be  applied 
during  the  twenty-four  hours  succeeding  an  injection, 
since  the  first  effect  of  the  vaccine  may  be  a  temporary 
lowering  of  resistance.  Vaccines  are  of  little  value,  and, 
in  general,  are  contraindicated  in  very  acute  infections, 
particularly  in  those  which  are  accompanied  by  much 
systemic  intoxication,  for  in  such  cases  the  power  of  the 
tissues  to  produce  antibodies  is  already  taxed  to  the 
limit.  It  is  true,  nevertheless,  that  remarkably  bene- 
ficial results  have  occasionally  followed  their  use  in  such 
acute  conditions  as  malignant  endocarditis,  but  here 
they  should  be  tried  with  extreme  caution. 

Probably  best  results  are  obtained  in  staphylococcus 
infections,  although  pneumococcus,  streptococcus,  and 
colon  bacillus  infections  usually  respond  nicely.  Among 
clinical  conditions  which  have  been  treated  successfully 
with  vaccines  are  furunculosis,  acne  vulgaris,  infected 
operation-wounds,    pyelitis,    cystitis,    subacute    otitis 

media,  osteomyehtis,  infections  of  nasal  accessory  si- 

38 


594  PREPARATION   AND   USE    OF  VACCINES 

nuses,  etc.  Vaccine  treatment  of  the  mixed  infection  is 
doubtless  an  important  aid  in  tuberculosis  therapy, 
and  in  some  cases  the  result  is  brilliant.  When,  as  is 
common,  several  organisms  are  present  in  the  sputum, 
a  vaccine  is  made  from  each,  excepting  the  tubercle 
bacillus,  of  which  autogeneous  vaccines  are  not  used  in 
practice.  To  avoid  the  delay  and  consequent  loss  of 
virulence  entailed  by  study  and  isolation  of  the  several 
varieties,  many  workers  make  the  bacterial  suspension 
directly  from  the  primary  cultures.  The  resulting  vac- 
cines contain  all  strains  which  are  present  in  the  sputum 
in  approximately  the  same  relative  numbers.  Although 
open  to  criticism  from  a  scientific  standpoint,  this 
method  offers  decided  practical  advantages  in  many 
cases. 

V.  PROPHYLACTIC  USE  OF  VACCINES 

It  has  been  shown  that  vaccines  are  useful  in  prevent- 
ing as  well  as  curing  infections.  Their  value  has  been 
especially  demonstrated  in  typhoid  fever.  Three 
doses  of  about  500  million,  1000  million,  and  1000 
million  typhoid  bacilli,  respectively,  are  given  about 
seven  to  ten  days  apart.  Results  in  the  army,  where 
the  plan  has  been  tried  on  a  large  scale,  show  that 
such  vaccination  is  effective,  protecting  the  individual 
for  six  months  to  a  year,  or  longer. 

VL  TUBERCULINS 

Tuberculins  contain  the  products  of  tubercle  bacilli 
or  their  ground-up  bodies,  the  latter  class  being  prac- 
tically vaccines.  They  are  undoubtedly  of  great  value 
in  the  treatment  of  localized  tuberculosis,  particularly 


TUBERCULINS  595 

of  bones,  joints,  and  glands;  and  are  of  rather  indefinite 
though  certainly  real  value  in  chronic  pulmonary  tuber- 
culosis, especially  when  the  disease  is  quiescent.  The 
best  known  are  Koch's  old  tubercuHn  (T.  O.),  bouillon 
filtrate  (B.  F.),  triturate  .residue  (T.  R.),  bacillary 
emulsion  (B.  E.)  and  purified  tuberculin  (Endotin). 
There  seems  to  be  little  difference  in  the  actions  of  these, 
although  theoretically  T.  R.  should  immunize  against 
the  bacillus  and  B.  F.  against  its  toxic  products.  The 
choice  of  tuberculin  is  much  less  important  than  the 
method  of  administration.  The  making  of  autogenous 
tuberculins  is  impracticable,  hence  stock  preparations 
are  used  in  practice. 

Since  the  dose  is  exceedingly  minute,  the  tuberculin 
as  purchased  must  be  greatly  diluted  before  it  is  avail- 
able for  use.  A  convenient  plan  is  to  use  the  rubber- 
capped  bottles  of  sterile  salt  solution  described  for  vac- 
cines (see  p.  585),  adding  sufficient  tuberculin  to  give 
the  desired  strength,  together  with  o.i  c.c.  trikresol  to 
insure  sterility.  The  practitioner  should  bear  in  mind 
that  while  tuberculin  is  capable  of  good,  it  is  also  capable 
of  great  harm.  Everything  depends  upon  the  dosage 
and  plan  of  treatment.  Probably  a  safe  beginning  dose 
for  a  pulmonary  case  is  o.ooooi  mg. ;  for  gland  and 
bone  cases,  about  o.oooi  mg.  The  intervals  are  about 
one  week  or,  rarely,  three  days,  when  very  small  doses 
are  given.  The  dose  is  increased  steadily,  but  with  ex- 
treme caution;  and  should  be  diminished  or  temporarily 
omitted  at  the  first  indication  of  a  "reaction,"  of  which, 
in  general,  there  are  three  forms: 

[a)  General. — Elevation  of  temperature  (often  slight), 
headache,  malaise. 


0^-^ 


596  PREPARATION    AND    USE    OF  VACCINES 

{b)  Local. — Increase  of  local  symptoms,  amount  of 
sputum,  etc. 

(c)  Stick. — Inflamm.atory  reaction  at  site  of  injection. 

Treatment  is  usually  continued  until  a  maximum  dose 
of  I  mg.  is  reached,  the  course  extending  over  a  year 
or  more. 

VII.  TUBERCULIN   IN  DIAGNOSIS 

The  tissues  of  a  tuberculous  person  are  sensitized 
toward  tuberculin,  and  a  reaction  (see  preceding  section) 
occurs  when  any  but  the  most  minute  quantity  of  tuber- 
culin is  introduced  into  the  body.  Non-tuberculous 
persons  exhibit  no  such  reaction.  This  is  utilized  in 
the  diagnosis  of  obscure  forms  of  tuberculosis,  the  test 
being  applied  in  a  number  of  ways: 

1.  Hypodermic  Injection. — After  first  determining 
the  patient's  normal  temperature  variations,  Koch's  old 
tuberculin  is  used  in  successive  doses,  several  days  apart, 
of  o.ooi,  o.oi,  and  o.i  mg.  A  negative  result  with 
the  largest  amount  is  considered  final.  The  reaction 
is  manifested  by  fever  within  eight  to  twenty  hours  after 
the  injection.  A  rise  in  temperature  of  i°F.  is  generally 
accepted  as  positive.  The  method  involves  some  danger 
of  lighting  up  a  latent  process,  and  has  been  largely 
displaced  by  safer,  although  perhaps  less  reliable, 
methods. 

2.  Calmette's  Ophthalmo-reaction. — One  or  2  drops 
of  0.5  per  cent,  purified  old  tuberculin  are  instilled  into 
one  eye.  Tuberculin  ready  prepared  for  this  purpose  is 
on  the  market.  If  tuberculosis  exists  anywhere  in  the 
body,  a  conjunctivitis  is  induced  within  twelve  to 
twenty-four  hours.     This  generally  subsides  within  a 


TUBERCULIN   IN   DIAGNOSIS  597 

few  days.  The  method  is  now  rarely  used  since  it  is 
not  without  some,  though  slight,  risk  of  injury  to  the 
eye.  It  is  absolutely  contraindicated  in  the  presence 
of  any  form  of  ocular  disease.  A  second  instillation 
should  not  be  tried  in  the  same  eye. 

3.  More  Reaction. — A  50  per  cent,  ointment  of  old 
tuberculin  in  lanolin  is  rubbed  into  the  skin  of  the  abdo- 
men, a  piece  about  the  size  of  a  pea  being  required. 
Dermatitis,  which  appears  in  twenty-four  to  forty- 
eight  hours,  indicates  a  positive  reaction.  The  oint- 
ment can  be  purchased  ready  for  use. 

4.  Von  Pirquet's  Method. — This  is  the  most  widely 
used  of  the  tuberculin  tests.  Two  small  drops  of  old 
tuberculin  are  placed  on  the  skin  of  the  front  of  the 
forearm,  about  2  inches  apart,  and  the  skin  is  slightly 
scarified,  first  at  a  point  midway  between  them,  and 
then  through  each  of  the  drops.  A  convenient  scarifier 
is  a  piece  of  heavy  platinum  wire,  the  end  of  which  is 
hammered  to  a  chisel  edge.  A  wooden  toothpick  with 
a  chisel-shaped  end  is  also  convenient.  This  is  held  at 
right  angles  to  the  skin,  and  rotated  six  to  twelve  times 
with  just  sufficient  pressure  to  remove  the  epidermis 
without  drawing  blood.  In  about  ten  minutes  the 
excess  of  tuberculin  is  gently  wiped  away  with  cotton. 
No  bandage  is  necessary.  A  positive  reaction  is  shown 
by  the  appearance  in  twenty-four  to  forty-eight  hours 
of  a  papule  with  red  areola,  which  contrasts  markedly 
with  the  small  red  spot  left  by  the  control  scarification. 

These  tests  have  very  great  diagnostic  value  in  chil- 
dren, especially  those  under  three  years  of  age,  but  are 
often  misleading  in  adults,  positive  reactions  occurring 
in    many    apparently   healthy   individuals.     Negative 


598  PREPARATION   AND   USE    OF  VACCINES 

tests  are  very  helpful  in  deciding  against  the  existence 
of  tuberculosis. 

VIII.  CUTANEOUS  TEST  FOR  SYPHILIS 

Noguchi  has  prepared  a  substance  called  luetin, 
which  produces  a  cutaneous  reaction  in  syphilis  similar 
to  the  tuberculin  skin  reaction  in  tuberculosis.  Luetin 
consists  of  ground  cultures  of  Treponema  pallidum 
sterilized  and  preserved  with  trikresol.  It  can  be 
purchased  through  any  pharmacy. 

A  small  drop  (0.05  c.c.)  of  luetin  is  injected  into  the 
skin  (not  under  it)  of  one  arm.  A  similar  preparation 
without  the  treponema  is  injected  into  the  skin  of  the 
other  arm  as  a  control.  A  positive  reaction  usually 
begins  within  forty-eight  hours  and  consists  of  an  in- 
flammatory induration,  papule,  or  pustule.  It  is  some- 
times delayed  three  or  even  four  weeks. 

The  test  is  positive  in  late  secondary,  tertiary, 
latent,  and  hereditary  syphilis,  but  is  usually  absent 
in  primary  and  early  secondary  cases.  In  general 
paralysis  and  tabes  dorsalis  it  is  inconstant.  Com- 
pared with  the  Wassermann  reax:tion  it  is  more  con- 
stant in  terti'ary  and  latent  syphilis,  while  the  Wasser- 
mann is  more  constant  in  primary  and  secondary  cases. 
Unlike  the  Wassermann,  the  reaction  does  not  dis- 
appear with  treatment,  but  persists  probably  until  a 
complete  cure  is  effected. 

Recent  work  has  shown  that  luetin  will  produce  the 
typical  reaction  in  non-syphilitic  persons  who  are  tak- 
ing potassium  iodid  or  other  drugs  containing  iodin; 
also  that  syphilitic  persons  will  react  to  intradermal 
injections  of  agar  and  certain  other  substances  in 
practically  the  same  way  as  they  react  to  luetin. 


SCHICK    TEST    FOR    IMMUNITY    TO    DIPHTHERIA      599 

JX.  SCHICK  TEST  FOR  IMMUNITY  TO  DIPHTHERIA 

By  means  of  this  test,  which  was  introduced  by 
Schick  in  19 13,  it  is  possible  to  select  from  a  group 
of  individuals  those  who  are  immune  to  diphtheria 
by  virtue  of  natural  or  artificial  immunity.  In  an 
epidemic  of  diphtheria,  therefore,  it  is  of  very  great 
value  as  a  means  of  determining  who  shall  and  who 
shall  not  receive  prophylactic  injections  of  antitoxin. 
The  reaction  is  not  applicable  to  the  diagnosis  of 
diphtheria.  As  performed  by  Koplik  and  Unger 
the  test  is  as  follows: 

An  ordinary  hypodermic  needle  is  bent  at  a  slight 
angle  (about  170  degrees)  ^^  inch  from  the  tip  and  is 
mounted  in  a  handle  which  leaves  only  ^-i  inch  pro- 
jecting. To  make  the  test,  cleanse  an  area  upon  the 
skin  of  the  forearm,  and  encircle  the  forearm  with  the 
thumb  and  index-finger,  holding  the  skin  tense  between 
them.  Dip  the  needle  into  pure  undiluted  diphtheria 
toxin  and  immediately  insert  its  unshielded  quarter- 
inch  into,  not  through  the  skin,  the  bend  making  it  easy 
to  insert  intradermally.  No  injection  is  necessary,  as 
enough  of  the  toxin  adheres  to  the  needle. 

No  reaction  occurs  in  those  who  are  immune  to 
diphtheria.  In  those  who  are  not  immune  a  distinct 
red  spot  about  i  to  2  cm.  in  diameter  appears  at  the 
site  of  injection  within  eight  hours.  This  is  followed 
by  marked  induration,  reaching  its  height  in  about 
forty-eight  to  seventy-two  hours. 


CHAPTER  X 

SERODIAGNOSTIC  METHODS ' 
I.  IMMUNITY 

With  exception  of  the  last,  the  diagnostic  methods 
here  described  depend  on  one  or  another  law  of  im- 
munity. These  laws  are  customarily  described  in 
terms  of  Ehrlich's  side-chain  theory.  It  is  not  prac- 
ticable to  undertake  a  detailed  discussion  of  the  theory 
here,  and  I  shall,  accordingly,  confine  myself  to  such 
discussion  and  definition  of  the  bodies  concerned  as 
will  enable  the  reader  to  undertake  the  reactions  him- 
self with  a  reasonably  intelligent  conception  of  their 
mechanism. 

Acquired  immunity,  that  form  of  immunity  resulting 
from  an  attack  of  a  given  disease,  depends  upon  the 
formation  within  the  body,  under  the  influence  of  the 
disease-producing  agent,  or  "antigen,"  of  bodies 
possessing  the  power  to  neutralize  the  poisons  pro- 
duced by  the  antigen,  or  to  destroy  or  otherwise  affect 
the  antigen  itself.  Since  the  action  of  these  bodies  is 
specific  {i.e.,  they  act  only  on  the  particular  antigen 
whose  presence  has  led  to  their  production),  the 
search  for  them  may  be  resorted  to  for  diagnostic 
purposes   whenever    they   can   be   found   more   easily 

^  By  Ross  C.  Whitman,  B.A.,  M.D.,  Professor  of  Pathology  in  the 
University  of  Colorado. 

600 


IMMUNITY  6oi 

than  can  the  antigen  itself.  With  certain  exceptions, 
to  be  noted  later,  the  presence  of  one  or  other  of  these 
bodies  may  be  regarded  as  pathognomonic  of  the 
corresponding  disease. 

The  several  "immune  bodies"  act  by  means  of 
different  mechanisms,  by  virtue  of  which  they  may  be 
classified  in  three  groups — the  three  orders  of  receptors 
of  Ehrlich's  side-chain  theory.  With  the  first  group 
we  are  not  immediately  concerned. 

1.  Receptors  of  the  First  Order. — These  are  receptors 
which  serve  simply  as  connecting  links  between  the 
disease-producing  agent  (or,  rather,  of  its  toxin)  and  the 
tissues.  Under  the  influence  of  the  antigen  they  are 
produced  in  excess,  and  are  finally  set  free  in  the  circu- 
lation. Here  they  seize  upon  and,  so  to  speak,  satu- 
rate the  free  valence  of  the  antigen,  while  it  also  is  still 
free  in  the  blood  and  lymph,  in  such  a  way  as  to  leave 
the  latter  no  chemical  affinities  by  means  of  which  it 
may  combine  with  similar  bodies  still  in  relation  with 
the  cells.  The  antigen  is  thus  rendered  inert.  This 
order  of  receptors  includes  only  the  antitoxins;  for 
example,  those  of  diphtheria  and  tetanus. 

2.  Receptors  of  the  Second  Order. — These  have  a  com- 
bining group  similar  to  that  of  the  first  order,  and,  in 
addition,  a  group  possessing  a  ferment-like  action,  by 
means  of  which  the  characteristic  action  of  the  body  is 
effected.  The  ferment,  or  zymophore,  group  is  readily 
destroyed  by  heat,  so  that  serum  to  be  used  for  any  of 
the  purposes  included  in  the  group  must  not  be  heated. 
The  group  includes  the  agglutinins,  responsible  for  the 
several  applications  of  the  Widal  reaction;  the  precipi- 
tins, responsible  for  one  of  the  biologic  methods  to  be 


6o2  SERODIAGNOSTIC    METHODS 

described  later  for  the  forensic  identification  of  blood- 
stains; and  the  opsonins. 

3.  Receptors  of  the  Third  Order. — These  bodies  con- 
sist of  two  combining  affinities  only.  One  of  these  com- 
bines with  suitable  analogous  groups  of  the  antigen,  the 
other  combines  with  a  substance  which  is  called  com- 
plement because  it  "complements"  or  supplements  or 
completes  the  specific  action  of  the  immune  substance. 
Complement  is  normally  present  in  the  blood,  but  is 
unable  to  act  upon  the  antigen  without  the  mediation 
and  aid  of  the  immune  body.  The  latter  is,  therefore, 
called  the  amboceptor  or  'tween  body.  It  is  (relatively) 
thermostabile  and  keeps  practically  indefinitely  under 
suitable  conditions.  It  is  to  be  remembered  that  this  is 
the  specific  immune  substance  whose  presence  or  ab- 
sence is  indicative  of  the  presence  or  absence  of  the  cor- 
responding disease.  The  native,  normally  present  com- 
plement is  (relatively)  thermolabile,  being  destroyed 
in  a  few  minutes  by  a  temperature  of  54°  to  56°C., 
and  keeps  only  a  few  hours  under  the  best  conditions. 
It  is  non-specific,  and  within  certain  limits  the  comple- 
ment of  one  species  may  be  substituted  for  that  of 
another.  Thus,  in  the  Wassermann  reaction,  com- 
plement containing  fresh  serum  from  guinea-pigs  is 
usually  substituted  for  the  normally  present  com- 
plement of  the  patient's  serum,  after  the  latter  has 
been  destroyed. 

This  group  contains  the  lysins  and  the  bodies  respon- 
sible for  the  various  applications  of  the  complement- 
deviation  method  to  the  diagnosis  of  syphilis  (Was- 
sermann reaction),  gonorrhea,  typhoid  fever,  forensic 
identification  of  blood,  etc. 


APPARATUS 


603 


II.  APPARATUS 

Before  the  description  of  the  several  tests  is  taken  up, 
I  shall  give,  to  save  space,  a  list  of  general  equipment 
needed  for  such  work.  Special  apparatus  needed  for 
some  of  the  tests  will  be  mentioned  in  connection  with 
these. 

I.  Centrifuge. — While  the  usual  small  electric  or 
water-driven  instrument  can  be  employed,  a  larger 
machine,  capable  of  holding  4  or  8  tubes  of  about 
50-c.c.  capacity,  is  desirable. 


CD    CD     c^    <=>    o 
CD    <::>    c::>    o     o 

CD       CD      CD      CD       CD 
CD       CD      CD      CCIDCP      CD 
CD      CD      CD       CO  I      p     C 
o      c?      cr>       O      L^     CD 


4 


^       CD    CD 
CD       CD      C^ 

c:>     CD     o 

CD       O       C^       CD 
0       CD      CD3       CD 


^ 


Fig.  229.— Convenient  test-tube  rack  tor  serum  work. 


2.  Scales,  about  o.i  to  100  Gm.  capacity. 

3.  Microscope,  magnifying  50  to  750  diameters. 

4.  Incubator  at  37°C. 

5.  Water-bath,  to  be  regulated  as  required. 

6.  A  large  number  of  test-tubes,  about  120  by  16  mm. 

7.  Test-tube  racks  to  accommodate  the  above.  A 
double  row  of  holes  is  very  convenient.  Still  better  is  a 
special  rack  (Fig.  229),  made  of  copper  or  zinc,  with 
six  rows  of  holes,  six  to  each  row.    A  sheet  of  metal 


6o4  SERODIAGNOSTIC   METHODS 

midway  between  top  and  bottom  contains  holes  to  cor- 
respond, so  that  tubes  are  held  without  danger  of  tip- 
ping. The  rack  holds  tubes  enough  for  i8  Wassermann 
reactions,  if  but  one  antigen  is  used.  A  similar  rack, 
made  round,  and  with  the  two  lower  sheets  of  metal 
small  enough  to  go  through  the  circular  opening  of  the 
water-bath,  while  the  top  sheet  is  larger,  so  as  to  rest 
on  the  edge  of  the  opening,  is  also  very  convenient. 

8.  Volumetric  pipets,  o.i  c.c.  in  one-hundredths, 
and  lo  c.c.  in  one-tenths.  The  graduation  should  start 
near  the  point  where  the  emptying  of  the  pipet  is 
stopped  by  capillarity. 

9.  Capillary  pipets  (see  Fig.  228),  made  from  soft- 
glass  tubing,  as  described  on  page  561.  The  tube 
should  be  of  such  a  size  that  the  ordinary  medicine- 
dropper  nipple  will  fit  it  snugly.  Such  pipets  are  useful 
for  a  variety  of  purposes.  After  being  used  once  they 
should  be  thrown  away. 

10.  Glass  Capsules. — These  may  be  purchased  or, 
with  a  little  practice,  can  be  readily  prepared  from  the 
same  sort  of  tubing  by  drawing  out  a  piece  at  both  ends, 
and  sealing  in  the  flame.  If  desired,  one  end  may  be 
bent  over  to  form  a  hook  at  the  point  where  the  narrow- 
ing begins  (see  Figs.  230  and  231). 

11.  An  all-glass  syringe,  such  as  the  Luer,  about  5-c.c. 
capacity,  with  a  fairly  large  needle,  say  19  or  20  gage, 
preferably  of  platinum. 

III.  REACTIONS  BASED  UPON  IMMUNE  BODIES  OF 

THE  SECOND  ORDER 

A.  The  Widal  Reaction 

The  test  may  be  employed  for  the  diagnosis  of  a 
variety     of     infections,     e.g.,     typhoid,     paratyphoid, 


THE    WIDAL    REACTION 


60: 


bacillary  dysentery,  the  plague,  Asiatic  cholera,  epi- 
demic meningitis,  etc.  In  clinical  work  it  is  used  only 
for  the  diagnosis  of  typhoid  and  paratyphoid  infections. 

I.  Materials  Required. — The  following  especial 
equipment  is  needed : 

I.  A  homogeneous  suspension  of  the  organism  or 
organisms  suspected  of  causing  the  disease.  Such  sus- 
pensions may  be  purchased  from  the  manufacturers  of 


Fig.  230. — Method  of  obtaining  blood  in  a  Wright  capsule:  A, 
Filling  the  capsule;  the  long  arm  should  be  held  more  nearly  horizontal 
than  is  here  represented ;  B,  the  bulb  has  been  warmed  and  the  capillary 
end  sealed  in  the  flame;  C,  cooling  of  the  capsule  has  drawn  the  blood 
to  the  sealed  end;  D,  the  serum  has  separated,  and  the  top  of  the  capsule 
has  been  broken  off. 


biologic  preparations,  or  may  be  prepared  by  the  worker 
himself.  In  the  latter  case  twenty-four-hour-old  agar- 
slant  cultures  (preferably  attenuated  by  long-continued 
growth  on  culture-media)  should  be  washed  off  with 
normal  salt  solution  (0.85-0.9  per  cent,  sodium  chlorid), 
containing  either  0.5  per  cent,  phenol  or  o.i  per  cent, 
formalin,  and  shaken  until  the  suspension  is  as  uniform 
as  possible.  Dilute  by  adding  more  of  the  salt  solution 
until  the  suspension  is  but  sUghtly  milky,  and  preserve 


6o6  SERODIAGNOSTIC   METHODS 

in  the  ice-box.  Such  a  suspension  will  keep  for  months. 
Shake  thoroughly  before  using.  The  suspension  will 
settle  less  rapidly  if  lo  per  cent,  lactose  is  added  to  it. 
Suspensions  which  show  any  tendency  to  spontaneous 
agglutination  cannot,  of  course,  be  used. 

2.  Instead  of  the  suspension  of  killed  bacteria,  living 
young  cultures  (not  over  twenty-four  hours  old)  of  at- 
tenuated organisms  can  be  employed. 

3.  About  0.1  c.c.  of  the  patient's  serum.  This  may  be 
obtained  by  pricking  the  cleansed  finger  or  ear  rather 


Fig.  231. — A  satisfactory  glass  capsule  for  obtaining  small  quan- 
tities of  blood,  as  for  the  Widal  test.  The  straight  tube  (a)  is  more 
convenient  to  carry  in  the  hand-bag  than  is  Wright's  capsule.  It 
may  be  bent  as  shown  in  the  lower  figure  by  brief  application  of  a 
match  flame  at  the  bedside.  After  the  tube  is  filled  the  ends  may  be 
sealed  with  the  match  flame. 

deeply  and  collecting  the  blood  in  one  of  the  capsules 
above  mentioned,  as  is  indicated  in  Figs.  230  and  231. 
More  than  one  capsule  should  be  at  hand,  so  that  a  fresh 
-one  may  be  substituted  if  the  first  is  plugged  by  fibrin 
before  enough  blood  is  obtained. 

When  the  capsules  are  not  at  hand,  blood  may  be  ob- 
tained in  little  vials  such  as  can  be  made  by  breaking  off  the 
lower  3-^  inch  of  the  tubes  which  have  contained  peptoniz- 
ing powder.  Vials  in  which  hypodermic  tablets  are  sold  can 
be  used,  but  are  somewhat  too  narrow.     They  must,  of 


THE    WIDAL   REACTION  607 

course,  be  well  cleaned.  One  of  these  is  filled  to  a  depth  of 
about  ^  inch  from  a  puncture  in  the  ear,  and  is  then  set 
aside  for  a  few  hours.  When  the  clot  has  separated  it  is 
picked  out  with  a  needle,  leaving  the  serum. 

Sufficient  blood  may  also  be  collected  by  allowing 
drops  to  dry  on  glass  or  unglazed  paper  (without  heat- 
ing), to  be  afterward  macerated  in  water.  In  this  case, 
however,  dilutions  can  only  be  made  approximately. 

4.  Slides,    preferably    hollow    ground,    cover-glas' 
vaselin. 

2.  Methods. — Two  methods  of  performing  the  test 
will  be  described: 

(i)  Macroscopic  Method. — Separate  the  clot  and 
serum  in  the  capsule  by  centrifugation,  nick  the  wall  of 
the  capsule  a  short  distance  above  the  serum  with  a  file, 
and  break  the  capsule  at  this  point,  Pipet  off  the 
serum,  place  it  in  a  clean  test-tube,  and  add  9  volumes 
of  salt  solution.  Counting  the  drops  of  serum  as 
they  fall  from  the  capillary  pipet,  and  adding  nine  times 
the  number  of  drops  of  salt  solution  will  give  sufficiently 
accurate  dilution.  Now  place  a  number  of  very  small 
test-tubes  in  a  rack,  and  add  to  each  one  except  the 
first  0.5  c.c.  of  salt  solution  by  means  of  a  volumetric 
pipet.  Then  place  in  the  first  and  second  tubes  only 
0.5  c.c.  of  the  diluted  blood-serum.  Shake  the  second 
tube,  and  with  the  pipet  transfer  0.5  c.c.  to  the  third 
tube.  Shake  this  tube  and  transfer  0.5  c.c.  to  the 
fourth  tube,  and  so  on,  to  the  end.  Discard  0.5  c.c. 
from  the  last  tube.  One  tube,  to  serve  as  control, 
should  contain  only  0.5  c.c.  of  salt  solution,  without 
any  serum.  The  volumetric  pipet  should  be  thoroughly 
rinsed  out  with  salt  solution  after  each  transfer.     One 


6o8  SERODIAGNOSTIC    METHODS 

thus  arrives  at  a  series  of  dilutions  of  the  serum,  as 
follows:  i-io,  I-20,  1-40,  1-80,  1-160,  1-320,  1-640, 
etc.  Now  add  to  each  tube  a  like  amount  (0.3  to 
0.5  CO.)  of  the  suspension  of  killed  bacteria.  This 
doubles  the  dilution  of  the  serum  in  each  of  the  tubes. 
Mix  all  the  tubes  thoroughly  by  shaking,  and  place  the 
rack  in  a  moderately  warm  place  or  in  the  incubator 
for  eight  to  twelve  hours.  In  those  tubes  in  which  the 
reaction  is  positive  there  will  be  found  a  sediment 
consisting  of  agglutinated  bacteria  at  the  bottom  of 
the  test-tube,  with  a  clear  supernatant  fluid.  The 
control  tube  and  the  negative  tubes  will  be  cloudy  and 
without  sediment. 

Dead  cultures  of  typhoid  bacilli,  together  with  all  appara- 
tus necessary  for  performing  the  macroscopic  test,  are  put 
up  at  moderate  cost  by  various  firms  under  the  names  of 
typhoid  diagnosticum,  typhoid  agglutometer,  etc.  Full 
directions  accompany  these  outfits. 

Bass  and  Watkins  have  described  a  modification  of  the 
macroscopic  method  (using  very  concentrated  suspen- 
sions of  the  bacilli)  by  which  the  test  can  be  applied  at 
the  bedside.  Agglutination  occurs  within  a  few  minutes. 
The  apparatus  has  been  put  upon  the  market  by  Parke, 
Davis  &  Co. 

(2)  Microscopic  Method. — Arrange  a  series  of  dilu- 
tions of  the  blood-serum  as  above,  or,  if  dried  blood  is 
used,  macerate  the  dried  clot  with  salt  solution  or  tap- 
water.  In  the  latter  case,  unless  the  size  of  the  original 
drop  of  blood  is  known,  the  color  is  the  only  guide  as  to 
the  degree  of  dilution.  A  light  amber  color  will  roughly 
correspond  to  a  dilution  of  1-50.  From  such  a  dilution 
others  can  be  prepared.     On  the  center  of  each   of 


THE    WIDAL    REACTION  609 

several  clean  cover-glasses  place  a  loopful  of  each  of  the 
several  dilutions,  employing  a  platinum  loop  of  about 
2  mm.  .  diameter.  With  the  same  loop  add  to  each 
droplet  of  diluted  serum  a  loop  from  a  twelve-  to 
twenty-four-hour-old  bouillon  culture  of  the  organism 
in  question,  or  of  a  suspension  in  salt  solution  prepared 
from  a  young  agar-slant  culture.  This  doubles  the 
dilution  of  serum  in  each  case.  One  cover-glass  con- 
taining no  serum  should  be  prepared  to  serve  as  a 
control.  Press  over  each  cover-glass  a  hollow-ground 
slide  previously  ringed  with  vaselin.  Turn  the 
slide  over  so  as  to  bring  the  cover-glass  on  top.  Dry- 
ing is  prevented  and  the  cover-glass  held  in  place  by 
the  vaselin. 

When  hollow-ground  slides  are  not  at  hand,  a  drop  each 
of  the  diluted  serum  and  the  bacterial  suspension  may  be 
placed  in  the  center  of  a  heavy  ring  of  vaselin  on  an  ordinary 
slide  and  a  cover-glass  applied  to  this.  Vaselin  containing 
an  antiseptic  must  not,  of  course,  be  used  for  this  purpose. 

Place  the  slides  in  a  moderately  warm  place  or  in  the 
incubator  at  37°C.  for  two  hours.  Examine  under  the 
oil-immersion  lens  or,  better,  the  high-power  dry  lens 
of  the  microscope,  using  very  subdued  light.  Yellow 
(artificial)  light  gives  a  clearer  view  than  does  white 
light.  In  the  negative  slides  and  in  the  control  the 
organism  will  be  found  moving  freely  (if  a  motile 
species)  and  not  clumped;  while  in  the  positive  slides  the 
organisms  are  found  motionless  and  gathered  in 
tangled  masses  and  balls,  i.e.,  they  are  agglutinated 
(Fig.  232).  Pseudoreactions,  in  which  there  are  a  few 
small  clumps  of  organisms  whose  motion  is  not  entirely 


6lO  SERODIAGNOSTIC   METHODS 

lost,  together  with  many  freely  moving  organisms 
scattered  throughout  the  field,  should  not  mislead. 

Jagic  suggests  that,  after  agglutination  has  taken 
place,  a  drop  of  the  suspension  be  mixed  with  a  drop 
of  India-ink  and  spread  upon  a  slide.  In  this  way  a 
permanent  record  may  be  kept.  To  insure  sterility  the 
preparation  may  be  fixed  by  heat. 

3.  Interpretation  of  Results. — Experience  has  shown 
that  not  much  significance  attaches  to  reactions  occur- 


% 

i 

'          .^r-    , 

-      - 

^ 

•' '  "^^  '■ 

T 

/ 

',       y 

tj. 

A 

^':>>^ 

t 

^ 

k 

Fig.  232. — Showing  clumping  of  typhoid  bacilli  in  the  Widal  reac- 
tion. At  one  point  a  crenated  red  blood-corpuscle  is  seen  (Wright  and 
Brown). 


ring  in  two  hours  with  dilutions  of  serum  less  than 
1-75  or  i-ioo.  With  killed  organisms  the  dilution  may 
be  somewhat  lower  than  when  living  organisms  are  em- 
ployed. On  the  other  hand,  recently  isolated  virulent 
cultures  are,  in  general,  more  resistant  to  agglutination 
than  old  attenuated  ones.  A  number  of  other  disease 
conditions  may  give  rise  to  a  positive  reaction  with  the 
typhoid  bacillus,  notably  infections  with  closely  related 


BIOLOGIC  INDENTIFICATION  OF  UNKNOWN  PROTEINS    6ll 

organisms,  such  as  the  colon  bacillus.  (In  such  cases, 
if  tests  are  made  with  several  species,  the  species  agglu- 
tinated in  the  highest  serum  dilution  may  generally,  but 
not  always,  be  regarded  as  the  cause  of  the  infection.) 
Agglutination  of  tx-phoid  bacilli  may  also,  though  rarely, 
occur  in  diseases  of  the  liver,  particularly  those  accom- 
panied by  jaundice,  and  in  pneumococcus  infections. 
The  Widal  test  is  of  no  value  if  the  patient  has  re- 
ceived anti-typhoid  vaccination  within  several  years 
previously. 

In  typhoid  the  average  time  of  first  appearance  of 
the  reaction  in  the  dilutions  above  recommended  is  the 
fourteenth  to  the  fifteenth  day  of  actual  disease,  which 
corresponds  roughly  to  the  eighth  or  tenth  day  of  ap- 
parent disease.  In  doubtful  cases  the  test  should  be 
repeated  at  frequent  intervals,  and  no  disappointment 
should  be  felt  if,  as  sometimes  (though  rarely)  happens, 
the  reaction  does  not  appear  until  the  twentieth  to  the 
twenty-fifth  day  of  the  disease.  It  is  evident,  there- 
fore, that  its  value  for  early  diagnosis  is  much  less 
than  that  of  the  blood-culture  (see  p.  346).  After  the 
Widal  reaction  first  appears  it  remains  throughout  the 
whole  course  of  the  disease  and  often  persists  for  ^''ears. 

B.  Biologic  Identification  of  Unknown  Proteins 

This  includes  the  differentiation  of  human  and  animal 
blood,  detection  of  meat  adulteration,  etc.,  by  means  of 
the  precipitin  test  (method  of  Uhlenhuth). 

I.  Materials  Required. — The  following  equipment  is 
needed : 

I.  Blood-serum  of  an  animal  (rabbit)  highly  im- 
munized against  the  protein  to  be  determined.     Im- 


6l2  SERODIAGNOSTIC    METHODS 

munize  several  rabbits  by  several  intravenous  or  intra- 
peritoneal injections  of,  for  example,  human  blood,  or 
better,  blood-serum.  Placental  blood  may  be  used,  or 
the  blood  may  be  obtained  as  for  the  Wassermann 
reaction.  The  doses  should  be  2  or  3  c.c.  and  should  be 
given  at  four-  or  five-day  intervals.  After  the  fourth  or 
fifth  dose  draw  2  or  3  c.c.  of  blood  from  an  ear  vein,  sepa- 
rate the  serum,  and  determine  its  strength  as  follows: 

Prepare  dilutions  of  (in  this  case)  human  blood-serum 
in  the  proportions  i-iooo,  1-5000,  1-10,000,  1-20,000, 
etc.,  using  physiologic  salt  solution  as  a  diluent.  Place 
2  c.c.  each  of  the  several  dilutions  in  a  series  of  test- 
tubes.  To  each  tube  add  o.i  c.c.  of  the  rabbit's  serum, 
without  shaking.  A  distinct  cloud  should  appear  in  the 
lowest  dilution  (i-iooo)  within  a  minute  or  two,  rapidly 
deepening  to  a  heavy  flocculent  precipitate;  the  reaction 
develops  somewhat  more  slowly  in  the  higher  dilutions, 
but  no  reaction  is  significant  which  occurs  after  more 
than  twenty  minutes. 

If  the  titration  results  as  above  described,  anesthetize 
the  rabbit  while  it  is  in  a  fasting  condition,  as  otherwise 
the  serum  is  apt  to  be  opalescent;  remove  the  anterior 
breast  wall  under  aseptic  conditions;  take  out  the  left 
lung  and  open  the  heart,  so  as  to  allow  the  animal  to 
bleed  to  death  into  its  pleural  cavity.  Cover  the  body 
with  sterile  towels  wet  with  antiseptic  solution  After 
clotting  has  occurred,  pipet  the  serum  into  sterile  bot- 
tles, and  add  3^1 0  volume  of  5  per  cent,  carbolic  acid  as 
a  preservative.  If  the  serum  is  opalescent,  it  cannot 
be  used;  if  cloudy,  it  must  be  filtered  clear  through  a 
sterile  Berkefeld  filter.  Sometimes  the  cloudiness  can 
be  removed  by  simple  sedimentation.     The  titration 


BIOLOGIC  INDENTIFICATION  OF  UNKNOWN  PROTEINS   613 

above  described  should  be  repeated  and  verified  before 
the  serum  is  used  for  making  the  test  proper. 

Other  sera  immune  to  horse,  dog,  sheep,  beef,  fowl, 
etc.,  may,  of  course,  be  prepared  in  the  same  way. 

2.  A  solution  of  the  unknown  substance  in  physio- 
logic salt  solution.  The  stock  dilution  should  be  about 
i-iooo.  If  made  from  a  dried  clot  this  can  only  be 
approximate.     The  following  criteria  may  be  used: 

(a)  It  should  be  almost  completely  colorless  by  trans- 
mitted light. 

{h)  It  should  give  only  a  slight  cloudiness  when 
heated  with  a  little  nitric  acid. 

(c)  It  should,  nevertheless,  foam  freely  on  shaking. 
The  solution  must  be  made  perfectly  clear — by  filtration 
if  necessary. 

2.  Method. — Arrange  a  series  of  test-tubes  and 
charge  them  as  follows: 

Tube  No.  I — -2  c.c.  of  the  unknown  solution  (diluted  i-iooo)  plus  o.i  c.c. 
of  immune  serum. 

Tube  No.  2 — 2  c.c.  of  normal  salt  solution  plus  0.1  c.c.  of  immune  serum. 

Tube  No.  3 — 2  c.c.  of  a  i-iooo  dilution  of  known  serum  of  the  species 
corresponding  to  that  suspected  to  be  present  in  the 
unknown  material  plus  o.i  c.c.  of  immune  serum. 

Tube  No.  4 — 2  c.c.  of  a  i-iooo  dilution  of  a  serum  from  a  species  differ- 
ent from  that  suspected  to  be  present  in  the  unknown 
material  plus  0.1  c.c.  of  immune  serum. 

Tube  No.  5 — 2  c.c.  of  the  unknown  solution  alone. 

When  the  first  and  third  tubes  give  a  positive  reac- 
tion, as  above  defined,  and  all  the  others  a  negative 
reaction,  the  presence  of  the  protein  of  the  species 
tested  for  is  established.  It  must  be  remembered  that 
shaking  must  not  he  employed.  When  only  limited 
amounts  of  material  are  available,  the  test  can  be  made 
by  contact  in  capillary  tubes. 


6l4  SERODIAGNOSTIC   METHODS 

Meat  adiilteration  may  be  recognized  by  the  same 
method.  Usually  it  is  a  question  of  horse  flesh  sold 
as  beef  or  as  sausage.  Remove  about  30  Gm.  of  the 
meat  from  the  deeper  portions  of  the  specimen  with  a 
clean  sterile  knife,  free  as  much  as  possible  from  fat, 
chop  up  on  a  clean  board,  and,  if  salted,  extract  several 
times  in  the  course  of  ten  minutes  with  sterile  distilled 
water.  Cover  the  30  Gm.  of  freshened  chopped  meat 
with  about  50  c.c.  of  0.85  per  cent,  salt  solution,  and 
allow  it  to  stand  three  hours  at  room-temperature  or 
over  night  in  the  ice-box.  Pipet  off  the  supernatant 
fluid,  and  clarify  and  dilute  for  use  according  to  the 
criteria  given  above  for  preparing  extracts  of  the  un- 
known substance.  If  acid  to  litmus,  it  is  to  be  neutral- 
ized before  use  by  adding  an  excess  of  an  insoluble 
alkali,  such  as  magnesium  oxid,  and  filtering. 

The  immune  serum  is  prepared  as  above  by  injecting 
rabbits  with,  in  this  case,  horse-serum.  It  must  have  a 
titer  of  at  least  1-20,000.  That  is,  it  must  give  a  reac- 
tion with  the  homologous  serum  in  a  dilution  of  the 
latter  of  that  degree. 

3.  Interpretation  of  Results. — These  reactions  are 
very  closely  specific,  and  are  fully  established  for  foren- 
sic purposes.  Doubt  can  only  arise  as  between  the  pro- 
teins of  very  closely  related  species,  and  this  can  be  practi- 
cally always  removed  by  the  use  of  adequate  controls. 

The  "Typhoid  Diagnosticum"  of  Ficker  is  based  on 
the  same  principle.  A  filtrate  of  an  autolyzed  culture 
is  used  instead  of  the  suspension  of  killed  typhoid 
bacilli  described  on  page  605.  The  dilutions  are  pre- 
pared as  for  the  Widal  test,  but  a  positive  reaction  is 
indicated  by  a  precipitate  instead  of  by  agglutination. 


OPSONINS  615 

C.  Opsonins 

That  phagocytosis  plays  an  important  part  in  the 
body's  resistance  to  bacterial  invasion  has  long  been 
recognized.  According  to  Metchnikoff,  this  property  of 
leukocytes  resides  entirely  within  themselves,  depending 
upon  their  own  vital  activity.  The  studies  of  Wright 
and  Douglas,  upon  the  contrary,  indicate  that  the 
leukocytes  are  impotent  in  themselves,  and  can  ingest 
bacteria  only  in  the  presence  of  certain  substances  which 
exist  in  the  blood-plasma.  These  substances  have  been 
named  opsonins.  Their  nature  is  undetermined. 
They  probably  act  by  uniting  with  the  bacteria,  thus 
preparing  them  for  ingestion  by  the  leukocytes;  but  they 
do  not  cause  death  of  the  bacteria,  nor  produce  any 
appreciable  morphologic  change.  They  appear  to  be 
more  or  less  specific,  a  separate  opsonin  being  necessary 
for  phagocytosis  of  each  species  of  bacteria.  There  are, 
moreover,  opsonins  for  other  formed  elements — red 
blood-corpuscles,  for  example.  It  has  been  shown  that 
the  quantity  of  opsonins  in  the  blood  can  be  greatly  in- 
creased by  inoculation  with  dead  bacteria. 

To  measure  the  amount  of  any  particular  opsonin  in 
the  blood  Wright  has  devised  a  method  which  involves 
many  ingenious  and  delicate  technical  procedures. 
Much  skill,  such  as  is  attained  only  after  considerable 
training  in  laboratory  technic,  is  requisite,  and  there 
are  many  sources  of  error.  It  is,  therefore,  beyond  the 
province  of  this  work  to  recount  the  method  in  detail. 
In  a  general  way  it  consists  in :  {a)  Preparing  a  mixture 
of  equal  parts  of  the  patient's  blood-serum,  a  suspension 
of  the  specific  micro-organism,  and  a  suspension  of 
washed    leukocytes;  {b)  preparing   a  similar  mixture, 


6l6  SERODI AGNOSTIC    METHODS 

using  serum  of  a  normal  person;  (c)  incubating  both 
mixtures  for  a  definite  length  of  time;  and  (d)  making 
smears  from  each,  staining,  and  examining  with  an  oil- 
immersion  objective.  The  number  of  bacteria  which 
have  been  taken  up  by  a  definite  number  of  leukocytes 
is  counted,  and  the  average  number  of  bacteria  per 
leukocyte  is  calculated;  this  gives  the  "phagocytic 
index."  The  phagocytic  index  of  the  blood  under 
investigation,  divided  by  that  of  the  normal  blood,  gives 
the  opsonic  index  of  the  former,  the  opsonic  index  of  the 
normal  blood  being  taken  as  i.  Simon  regards  the 
percentage  of  leukocytes  which  have  ingested  bacteria 
as  a  more  accurate  measurement  of  the  amount  of 
opsonins  than  the  number  of  bacteria  ingested,  because 
the  bacteria  are  apt  to  adhere  and  be  taken  in  in  clumps. 
Because  of  its  simplicity  the  clinical  laboratory 
worker  will  prefer  some  modification  of  the  Leish- 
man  method,  which  uses  the  patient's  own  leukocytes. 
It  is,  perhaps,  as  accurate  as  the  original  method  of 
Wright,  although  variations  in  the  leukocyte  count  have 
been  shown  to  affect  the  result.  Two  pipets  like  those 
shown  in  Fig.  228  are  used. 

1.  Make  a  suspension  of  the  specific  organism  by  mixing 
a  loopful  of  a  young  agar  culture  with  i  c.c.  of  a  solution  con- 
taining I  per  cent,  sodium  citrate  and  0.85  per  cent,  sodium 
chlorid.  Thoroughly  break  up  all  clumps  by  sucking  the 
fluid  in  and  forcing  it  out  of  one  of  the  capillary  pipets  held 
vertically  against  the  bottom  of  the  watch-glass. 

2.  Puncture  the  patient's  ear,  wipe  off  the  first  drop  of 
blood,  and  from  the  second  draw  blood  into  the  other  pipet 
to  the  wax-pencil  mark,  let  in  a  bubble  of  air,  and  draw  in 
the  same  amount  of  bacterial  suspension. 


OPSONINS  617 

3.  Mix  upon  a  slide  by  drawing  in  and  forcing  out  of  the 
pipet. 

4.  Draw  the  mixture  high  up  in  the  pipet,  seal  the  tip 
in  the  flame,  and  place  in  the  incubator  for  fifteen  minutes. 

5.  Repeat  steps  (2),  (3)  and  (4)  with  the  blood  of  a 
normal  person. 

6.  After  incubation,  break  off  the  tip  of  the  pipet,  mix 
the  blood-bacteria  mixture,  and  spread  films  on  slides. 

7.  Stain  with  Wright's  blood-stain  or  carbol-thionin. 

8.  With  an  oil-immersion  lens  count  the  bacteria  which 
have  been  taken  in  by  100  leukocytes,  and  calculate  the 
average  number  per  leukocyte.  Divide  the  average  for  the 
patient  by  the  average  for  the  normal  person.  This  gives 
the  opsonic  index.  If  in  the  patient's  blood  there  was  an 
average  of  4  bacteria  per  leukocyte,  and  in  the  normal  blood 
5  bacteria  per  leukocyte,  the  opsonic  index  would  be  ^^ 
or  0.8. 

Wright  and  his  followers  regarded  the  opsonic  index 
as  an  index  of  the  power  of  the  body  to  combat  bacterial 
invasion.  They  claimed  very  great  practical  im- 
portance for  it  as  an  aid  to  diagnosis  and  as  a  guide  to 
treatment  by  the  vaccine  method.  This  method  of 
treatment  consists  in  increasing  the  amount  of  pro- 
tective substances  in  the  blood  by  injections  of  normal 
salt  suspensions  of  dead  bacteria  of  the  same  species  as 
that  which  has  caused  and  is  maintaining  the  morbid 
process,  these  bacterial  suspensions  being  called  "vac- 
cines." Vaccine  Therapy  (Chapter  IX)  has  probably 
taken  a  permanent  place  among  our  methods  of  treat- 
ment of  bacterial  infections,  particularly  of  those  which 
are  strictly  local,  but  the  opsonic  index  is  now  little 
used  either  as  a  measure  of  resisting  power  or  as  an  aid 
to  diagnosis  and  guide  to  treatment. 


6l8  SERODIAGNOSTIC    METHODS 

IV.  REACTIONS  BASED  UPON  IMMUNE  BODIES  OF 
THE  THIRD  ORDER 

The  reactions  of  this  group  comprise  the  various 
applications  of  the  Wassermann  reaction  or,  more 
properly,  of  the  Bordet-Gengou  phenomenon  of  comple- 
ment-fixation or  deviation.  Since  the  reaction  involves 
three  active  substances — viz.,  antigen  (the  substance 
inducing  the  immune  reaction) ;  the  specific  amboceptor, 
or  immune  substance;  and  the  non-specific  complement 
— it  is  possible  to  so  adjust  matters  that,  any  two 
factors  being  known,  the  third  may  be  determined 
either  qualitatively  or  (roughly)  quantitatively.  Prac- 
tically, the  method  is  employed  chiefly  for  determining 
the  presence  of  the  middle  term,  or  amboceptor.  It 
may  be  applied  to  the  diagnosis  of  any  disease  the 
antigen  of  which  is  knowm  and  which  can  be  obtained 
in  suitable  form.  This  includes  syphilis,  gonorrhea, 
tuberculosis,  echinococcus  and  cysticercus  diseases, 
trichiniasis,  tj'phoid  fever,  and  pneumococcus,  meningo- 
coccus and  staphylococcus  infections,  etc.  In  several 
of  these,  other  and  simpler  methods  are,  however,  avail- 
able. The  method  as  applied  to  the  first  three  diseases 
above  mentioned  is  given  below. 

The  method  employed  is  based  upon  the  fact  that  if 
suitable  quantities  of  antigen,  amboceptor  (i.e.,  patient's 
serum  containing  the  same),  and  complement  are  mixed 
together  and  warmed  gently  in  the  incubator,  a  sup- 
posedly chemical,  firm  union  of  the  three  takes  place. 
The  mere  fact  of  combining  in  this  way  produces, 
however,  no  visible  change  in  the  fluid.  It  is  necessary, 
therefore,  to  test  for  free  complement  by  adding  the 
two  other  units  of  another  immune  system  which  also 


COMPLEMENT   DEVIATION   TEST  619 

requires  the  presence  of  complement,  and  which  will 
produce  a  visible  reaction  if  free  complement  is  present. 
A  "hemolytic  system"  is  used  for  this  purpose.  The 
mixture  is  then  incubated  a  second  time.  If  the  three 
units  of  the  first  system  have  combined  (in  other  words, 
if  the  patient's  serum  contains  syphilis  antibody),  and 
not  otherwise,  the  complement  is  "fixed"  or  "deviated" 
during  the  first  incubation  period,  so  that  it  is  no  longer 
available  to  assist  in  completing  the  second  dnd  visible 
reaction  represented  by  the  hemolytic  system.  As  will 
be  seen  later,  an  elaborate  system  of  controls  is  needed. 

A.  Complement  Deviation  Test  for  Syphilis 
The  Wassermann  Reaction 

Of  the  many  modifications  of  the  Wassermann  reac- 
tion, but  one,  the  standard  form  of  the  reaction,  will  be 
given. 

I.  Materials  Required. — The  following  reagents  are 
needed : 

I.  Syphilitic  Antigen. — The  reaction,  originally  sup- 
posed to  depend  upon  the  presence  in  the  patient's 
serum  of  true  syphilis  antibodies,  is  now  known  to 
depend  instead  on  a  disorder  of  lipoid  metabolism  char- 
acterized by  the  presence  of  serum-foreign  lipoids 
in  the  serum.  Accordingly,  solutions  of  lipoids  from 
various  sources  can  be  used  for  the  test.  The  following 
may  be  recommended: 

(a)  Grind  or  chop  the  liver  and  spleen  of  a  syphilitic 
fetus.  Place  in  a  suitable  vessel  and  add  4  to  10  parts 
of  absolute  ethyl  alcohol.  (The  amount  of  alcohol 
varies  in  the  hands  of  diff^erent  workers.)  Extract  for 
three  or  four  days  in  the  incubator  or  for  one  to  two 


620  SERODIAGNOSTIC    METHODS 

weeks  at  room-temperature,  with  frequent  vigorous 
shakings.  Filter  through  paper.  The  filtrate  con- 
stitutes the  stock  solution,  which  is  diluted  with  salt 
solution  for  use,  as  described  later. 

(b)  Grind  in  a  mortar  with  quartz  sand  one  or  more 
guinea-pig  hearts,  previously  weighed,  place  in  a  suit- 
able receiver,  and  add  lo  c.c.  of  absolute  alcohol  for 
each  gram  of  heart  tissue.  Complete  the  preparation 
as  above.  This  solution  can  be  purchased  from  the 
various  biologic  houses 

(c)  The  "fortified"  or  cholesterinized  antigen  of 
Swift  is  prepared  from  (b)  by  dividing  a  given  lot  in 
half,  saturating  one  of  the  halves  with  Merck's  cho- 
lesterin  in  the  incubator,  placing  in  water  at  i5°C. 
for  two  hours,  and  finally  filtering  and  mixing  with  the 
other  half. 

2.  Antisheep  Amboceptor. — This  can  now  be  obtained 
so  readily  in  the  market  that  the  somewhat  elaborate 
method  of  preparation  may  be  omitted  here. 

3.  Sheep's  Red  Blood-cells. — Where  a  slaughter-house 
is  available,  it  constitutes  the  most  convenient  source  of 
supply.  A  sterile  bottle  (about  loo-c.c.  capacity),  con- 
taining some  glass  beads,  bits  of  glass  rod,  or  steel 
shavings,  is  carried  to  the  slaughter-house.  After  the 
first  gush  of  blood  from  the  slaughtered  animal  has 
cleansed  the  wound,  the  bottle  is  filled  not  quite  full 
with  blood.  It  is  then  stoppered  and  the  bottle  kept 
in  motion  for  ten  or  fifteen  minutes  or  until  defibrination 
is  complete.  For  use,  "wash"  the  cells  thoroughly  free 
from  serum  by  filling  centrifuge  tubes  about  one- 
quarter  full  of  defibrinated  blood,  and  adding  0.9  per 
cent,  sodium  chlorid  solution  to  the  top.     Centrifugate 


COMPLEMENT    DEVIATION    TEST  62 1 

thoroughly  and  pipet  off  the  supernatant  fluid.  Again 
fill  with  salt  solution,  mix,  centrifugate,  and  remove  the 
supernatant  fluid.  Repeat  at  least  three  times.  Finally, 
prepare  a  5  per  cent,  emulsion  by  adding  i  volume  of 
the  cells,  thoroughly  packed  by  centrifugation,  to  19 
volumes  of  salt  solution.  This  is  the  standard  against 
which  the  strength  of  all  other  solutions  is  measured 
or  titrated,  as  described  below. 

The  following  method,  a  modification  by  W.  W. 
Williams  of  that  of  Rous  and  Turner,  furnishes  cells 
which  remain  serviceable  for  about  two  weeks — a 
much  longer  period  than  do  those  preserved  by  the 
customary  method. 

Place  in  a  quart  Mason  jar  the  following: 

Granulated  sugar ii.o  Gm.; 

Sodium  citrate  (Merck) 8.0  Gm.; 

Gelatin,  bacteriological 2.0  Gm.; 

Distilled  water 35°  o  c.c; 

Liquefied  phenol 1.5  c.c. 

Sterilize  jar  and  contents  in  the  autoclave  at  15  pounds 
for  ten  minutes.  At  the  abattoir  fill  to  within  about 
an  inch  of  the  top  with  fresh  sheep's  blood.  Preserve 
in  the  ice-box.  Small  portions  may  be  removed  and 
washed  for  use  as  required. 

4.  Complement. — Stun  a  fasting  guinea-pig  by  a  blow 
at  the  base  of  the  skull,  cut  the  throat,  and  collect  the 
blood  in  a  clean,  dry  dish.  The  complement  will  be 
destroyed  if,  by  cutting  the  esophagus,  stomach  con- 
tents become  mixed  with  the  blood.  The  serum  may 
be  allowed  to  separate  spontaneously  over  night  in  the 
ice-bo"x,  or  be  separated  just  before  use  by  centrifuga- 
tion. Serum  more  than  twenty-four  hours  old  is  worthless 
as  complement. 


622  SERODIAGNOSTIC   METHODS 

5.  Patient's  Serum. — About  5  c.c,  of  blood  will  suffice. 
A  convenient  method  consists  in  applying  an  Esmarch 
bandage  to  the  upper  arm,  after  cleansing  the  flexor 
surface  of  the  elbow  with  alcohol  or  tincture  of  iodin. 
If  the  patient  opens  and  closes  the  fist  vigorously  a  few 
times  the  veins  become  more  prominent.  Insert  the 
needle  of  the  syringe  above  described  above  or  along- 
side the  vein  and  at  an  acute  angle  to  the  skin  surface. 
Once  through  the  skin,  a  httle  practice  will  enable  one 
to  quickly  find  the  way  into  the  vein.  Slow  withdrawal 
of  the  plunger  will  quickly  fill  the  syringe.  If  the  vein 
is  a  large  one  the  blood  will  flow  into  the  syringe,  driv- 
ing the  plunger  ahead  of  it.  Remove  the  bandage  be- 
fore withdrawing  the  syringe  to  avoid  a  hematoma. 
Withdraw  the  needle  quickly,  and  have  the  patient 
or  an  assistant  apply  fairly  firm  pressure  over  the 
punctured  vein  for  a  minute  or  two.  In  the  meantime 
empty  the  syringe  into  a  scrupulously  clean  test- 
tube,  and  immediately  wash  out  the  syringe  and 
needle  thoroughly  with  water,  followed,  especially  if 
the  needle  is  of  steel,  by  alcohol.  If  blood  is  given 
time  to  clot  in  the  needle  or  syringe  the  instrument  is 
practically  ruined.  The  needle  should,  of  course,  be 
sterilized  by  boiling  before  use.  The  syringe  should 
be  clean  and  dry  (as  otherwise  hemolysis  will  take 
place),  but  need  not  be  sterilized. 

After  an  hour  or  two,  separate  the  clot,  if  necessary, 
from  the  test-tube  wall  with  a  clean  wire,  and  either 
com.plete  the  separation  of  the  serum  at  once  by 
centrifugation  or  place  in  the  ice-box  over  night. 
Transfer  the  serum  with  a  capillary  pipet  to  a  second 
clean  test-tube. 


COMPLEMENT   DEVIATION    TEST  623 

Before  the  test  is  made  the  serum  is  "inactivated" 
{i.e.,  the  native  complement  present  is  destroyed)  by 
immersing  the  tube  for  half  an  hour  in  the  water-bath 
at  55°  to  56°C. 

Unless  a  considerable  number  of  sera  are  to  be  exam- 
ined simultaneously,  known  positive  and  known 
negative  control  sera  must  be  prepared  in  the  same  way. 

2.  The  Titrations. — The  strength  of  the  complement 
and  antisheep  amboceptor  must  be  determined  on  each 
occasion  of  its  use.  The  antigen  must  be  titrated  every 
few  weeks. 

(i)  Titration  of  the  Complement. — The  complement 
may  be  used  undiluted  or  in  varying  dilutions  of  from 
40  to  10  per  cent.  The  greater  the  dilution,  of  course, 
the  greater  the  accuracy  with  which  it  can  be  titrated. 
Assuming  that  it  is  to  be  used  in  a  40  per  cent,  dilution 
(i  part  of  complement  serum  to  i^^  parts  of  salt  solu- 
tion) ,  arrange  a  series  of  test-tubes  somewhat  as  follows : 

Tube  No.  I — 0.02  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 
blood-cells  and  iH  units  amboceptor. ^ 
^  One  unit  of  amboceptor  is  the  amount  required  to  bring  about  solu- 
tion of  I  c.c.  of  the  5  per  cent,  red  cell  emulsion,  in  the  presence  of  i  unit 
of  complement,  in  one  hour  at  incubator  temperature.  In  the  same  way 
I  unit  of  complement  is  the  amount  required  to  bring  about  solution  in 
the  presence  of  one  unit  of  amboceptor  under  the  same  conditions.  In 
the  above  experiment  the  i}^  units  of  amboceptor  is  only  approximate. 
It  is  assumed  that  the  worker  has  purchased  amboceptor  in  i-c.c.  vials, 
guaranteed  to  contain  1000  units,  and  actually  containing  a  slight  excess 
over  that  amount.  For  use  this  is  diluted  with  100  parts  of  salt  solution; 
0.1  c.c.  will  then  contain  something  over  i  unit.  On  the  first  occasion  of 
its  use,  0.15  c.c.  may  be  accepted  for  titration  purposes,  the  aim  being 
to  use  a  moderate  excess  to  allow  for  the  chance  of  deterioration  and 
slight  variaf  ions  in  the  strength  of  the  blood  emulsion.  On  each  later 
occasion  the  approximate  value  is  known  from  the  last  previous  titra- 
tion. Amboceptor  dilutions  keep  well  in  the  ice-box,  but  may  undergo 
a  very  abrupt  deterioration  at  the  end  of  about  six  months. 


624  SERODIAGNOSTIC    METHODS 

Tube  No.  2 — 0.04  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  i^i  units  amboceptor. 
Tube  No.  3 — 0.06  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  iji  units  amboceptor. 
Tube  No.  4 — 0.08  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  iM  units  amboceptor. 
Tube  No.  5 — o.io  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  i}-i  units  amboceptor. 
Tube  No.  6 — 0.12  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  i}i  units  amboceptor. 

Make  up  all  tubes  to  a  like  volume  (1.5  or  2  c.c). 
Mix  thoroughly  by  gentle  shaking,  and  place  in  the 
incubator  (preferably  standing  in  a  dish  of  water,  since 
this  insures  rapid  and  uniform  heating  to  incubator 
temperature)  at  37°C.  for  one  hour.  The  tube  contain- 
ing the  smallest  amount  of  complement  which  shows 
complete  solution  of  the  red  cells  (the  solution  bright 
red,  perfectly  clear,  and  free  from  sediment)  contains 
one  unit  of  complement.  Twice  this  amount  is  used 
in  making  the  test  proper,  to  allow  for  the  rapid 
deterioration  which  takes  place  and  for  the  small 
amount  of  complement  directly  absorbed  by  the 
antigen. 

(2)  Titration  of  the  Amboceptor. — Arrange  tubes  as 
follows : 

Red  cells  Amboceptor 

Tube  Complement        (s  per  cent.)    (i -100  dilution) 

No.  1 1 3-2  units'  1 .0  c.c.  0.06  c.c 

No.  2 1 3-2  units  i.o  c.c.  0.08  c.c. 

No.  3 1 3-2  units  1.0  c.c.  o.io  c.c. 

No.  4 1 32  units  1.0  c.c.  0.12  c.c. 

No.  s i}'i  units  i  o  c.c.  0.14  c.c. 

No.- 6 13-^  units  i.o  c.c.  0.16  c.c. 

Bring  all  tubes  to  a  like  volume,  mix,  and  incubate  for 
one  hour.     The  tube  containing  the  smallest  amount 
1  As  determined  in  the  previous  titration. 


COMPLEMENT    DEVIATION    TEST  625 

of  amboceptor  which  causes  complete  hemolysis  contains 
one  unit.     Tu^o  units  are  used  for  the  test  proper. 

(3)  Titration  of  the  Antigen. — The  stock  solution  is 
to  be  diluted  freshly  for  use  with  salt  solution.  This 
makes  a  milky  fluid.  The  amount  of  dilution  will 
vary  with  the  strength  of  the  stock  solution  as  deter- 
mined by  the  following  tests.  For  the  latter  a  lo  per 
cent,  dilution  may  be  employed. 

Arrange  test-tubes  as  follows: 

Antigen  Red  cells 

Tube  .  (10  per  cept.)  (s  per  cent.) 

No.  I O.I  c.c.  i.o  c.c. 

No.  2 0.2  c.c.  1.0  c.c. 

No.  3 0.3  c.c.  1 .0  c.c. 

No.  4 0.4  c.c.  1.0  c.c. 

No.  5 0.5  c.c.  1 .0  c.c. 

No.  6 0.6  c.c.  1.0  c.c. 

Bring  all  tubes  to  a  like  volume.  Mix  and  incubate. 
The  amount  used  in  making  the  test  proper  must  not  be 
more  than  one-half  the  smallest  amount  which  causes 
hemolysis  in  the  above.  A  modified  form  of  this  titra- 
tion is  repeated  each  time  the  antigen  is  used. 

At  the  same  time  with  the  above  arrange  six  test- 
tubes  as  follows: 

Antigen 
Tube  (10  per  cent.)  Complement 

No.  1 0.1  c.c.  2  units 

No.  2 0.2  c.c.  2  units 

No.  3 0.3  c.c.  2  units 

No.  4 0.4  c.c.  2  units 

No.  5 0.5  c.c.  2  units 

No.  6 0,6  c.c.  2  units 

Bring  all  tubes  to  a  like  volume,  mix,  and  incubate. 
Then  add  to  all  tubes  i  c.c.  of  5  per  cent,  red  cell  emul- 
sion and  2  units  amboceptor  solution.     Mix  and  reincu- 

40 


626  SEB0DIAG99OS11C  MEmODS 

bate.  H  the  antigen  b  '^aiitimmpicmauary"  it  wffl 
kEMofysB  m  one  or  move  of  tlK  tnb^  Hie 
«9edfar  tbe  test  proper  must  nataaxedaoR- 

Tke  amligitBL  ■nost  also  be  siuwii  to  react  witli  known 
puftili*^  seta,  and  the  amnimt  icquiicd  to  pmdasx  a 
irarlinn  drlfi  mined-  For  this  poiposc  an  abundant 
nifyly  oC  seram  fitont  a  patient  widi  active  seoondaiy 
hjlibJBi  (si31  better,  from  several  sodi  patients)  is  ob- 
I  ■■!  d,  and  Ae  mmplrihr  irartion  carried  oat  as 
ilrsi  lilieil  bdov,  i  mtiliij  iiift  varyiog  amoonts  ni  the 
anllgM^  tiihiliiin^,  c^-,  o^Qtj  ouo6,  ouo8,  oj,  olI2,  OL14  cc, 
etc  Forthetealpmpei  an  amnnntBnsedin  thegreatest 
fwiwJiJr  cscess  of  that  aninnnt  which  ^ves  a  positire 
leactioMj  bat  winch  <«iMnilirs^,  ucvcitheiess,  with  the 

above  as  to  hemolytic  and 


L — ^i.  IHrty  g^baswaie 
for  most  of  the  emus, 
an  cnor  caused  by  a  sin^k 
a  handled  clean  ones.  The 
system  win  be  innnphrtely  destroyed  in  socfa 
a  tube;,  wiih  the  IftrBhnod  of  infteipielii^  the  lesolt  as 
a  poattivc  icaction,  or  as  dne  to  an  anticannplementaiy 
patient's  senon,  etc^  dtpenfing  on  ^diat  it  happens  to 
bensedfor.  G]as5waieneednotbesieiilei,botfluijl&e 
mkadmtdjf  deam.  Never  aDow  used  tnbes  to  stand  and 
Ajont.  ImaM&tcfy  the  wodc is  fini^ed  wa^  with 
so^  and  water,  fime  thorongld^  with  dean  wata 
iolowed  brjr  dOnte  (so  per  cent.)  mtoc  acid,  then 
soda  'Ml-tMM^  tibcn  sevenl  cJianges  of  disfined 
Place  m  a,  basket  and  dry  in  a  diy-air  strriKnT 


COMPLEMENT   DEVIATION   TEST  627 

if  one  is  available.  New  glassware  should  be  prepared 
for  use  in  the  same  way.  Tubes  may  be  kept  stored 
in  the  oven  or  set  aside  in  a  clean  dust-proof  cupboard. 
Tubes  used  for  this  purpose  should  never  be  used  for 
any  other.  Other  glassware  should  be  cleaned  with 
equal  care,  and  preserved  from  any  other  use. 

2.  The  patient's  serum  may  be  "anticomplementary," 
i.e.,  it  may  have  the  power  to  combine  with  or  absorb 
complement  in  the  absence  of  antigen.  Serum  which 
has  been  kept  too  long,  or  which  has  been  inactivated 
at  a  temperature  above  56 °C.,  is  apt  to  exhibit  this 
property.  The  anticomplementary  property  may  some- 
times be  made  to  disappear  by  renewed  inactivation. 
If  this  fails,  the  serum  must  be  discarded.  A  control  of 
this  property  is  included  in  setting  up  the  test; 

3.  The  antigen  may  become  anticomplementary  or 
hemolytic,  or  both.  When  this  happens  it  must  be  dis- 
carded. A  control  for  this  is  set  up  each  time  the 
antigen  is  used. 

4.  The  hemolytic  system  may  fail  to  function  for  a 
variety  of  reasons.  A  control  of  this  is  furnished  by 
the  titration  of  complement  and  amboceptor  above 
described. 

4.  The  Test  Proper. — After  all  preparations  have 
been  completed  and  the  titrations  satisfactorily  per- 
formed, one  proceeds  to  set  up  the  test  proper.  We 
may  suppose  that  we  are  dealing  with  at  least  three 
sera — viz.,  the  patient's  serum  and  known  positive  and 
known  negative  controls.  In  a  rack  having  two  rows 
of  holes  arrange  test-tubes  as  shown  on  page  629.  Mix 
by  gentle  shaking  and  place  in  the  incubator  for 
one   hour.     The   rack   should    stand    with    the    tubes 


628  SERODIAGNOSTIC    METHODS 

immersed  in  water  to  about  the  level  of  the  contents. 
Then  add  to  all  the  tubes  except  the  last  one,  already 
containing  blood-cells,  2  units  of  antisheep  amboceptor 
dilution  and  i  c.c.  of  5  per  cent,  sheep  red  cell  emulsion. 
Mix  as  before  and  incubate  for  two  hours.  The  tubes 
in  the  back  row  show  for  each  serum  tested  whether 
any  of  them  is  anticomplementary.  They  should  all 
show  complete  solution  of  the  red  cells.  The  last  two 
tubes  show  whether  the  antigen  is  hemolytic  or  anticom- 
plementary respectively.  The  first,  containing  com- 
plement, should  show  complete  hemolysis.  The  second, 
containing  only  antigen  and  red  cells,  should  show  no 
solution.  Assuming  that  these  controls  are  all  satis- 
factory, one  turns  to  the  tubes  in  the  front  row.  The 
known  positive  control  shows  no  solution  of  the  red 
cells,  the  complement  having  been  deviated  or  bound 
during  the  first  incubation,  and  hence  being  not  available 
for  the  reaction  with  the  red  cells.  For  the  opposite 
reason  the  known  negative  serum  will  show  complete 
solution.  The  unknown  serum  will  behave  like  the 
first  or  the  second,  according  as  it  is  positive  or  negative. 

It  is  apparent  that  an  excess  of  complement  may  con- 
vert a  positive  reaction  into  a  negative,  while  a  defi- 
ciency may  cause  a  negative  serum  to  behave  like  a 
(more  or  less)  positive  one.  It  is  to  avoid  this  contin- 
gency that  the  unknown  quantity  of  complement  present 
in  the  serum  of  the  patient  is  removed  by  inactivation, 
to  be  replaced  by  an  accurately  measured  amount  of 
guinea-pig  complement. 

With  cerebrospinal  fluid  the  reaction  is  carried  out 
in  the  same  way,  except  that  this  fluid  must  not  he  inac- 
tivated, and  is  used  in  larger  amounts.     When  enough 


COMPLEMENT   DEVIATION    TEST 


629 


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630  SERODIAGNOSTIC   METHODS 

fluid  is  available  the  test  should  be  set  up  so  as  to  give 
a  reading  for  0.4,  0.6,  0.8,  and  i  c.c.  When  economy 
of  material  is  necessary  a  reading  should  be  obtained 
for  0.5  and  i  c.c.  Further  modifications  may,  of 
course,  be  imposed  by  the  exigencies  of  the  case. 

Several  degrees  of  the  reaction  are  recognized  and  are 
customarily  indicated  as  follows: 

Complete  inhibition  (cells  intact  with  colorless 

supernatant  fluid),  +  +  +  +  or  4  +. 

Almost  complete  inhibition,  +  +  +  or  3  +. 

About  one-half  complete  inhibition,  ++  or  2  +. 

Slight  inhibition,  +  or  i  -f. 

No  inhibition.  o. 

5.  Interpretation  of  Results. — i .  Jaundice  and  marked 
alcoholism  may  convert  a  positive  reaction  into  a  nega- 
tive one. 

2.  Scarlet  fever,  leprosy,  active  malaria,  and  malig- 
nant tumors  may  cause  a  positive  reaction. 

3.  The  reaction  is  negative  in  primary  syphilis,  but 
becomes  rapidly  and  strongly  positive  as  the  general 
manifestations  of  the  disease  develop.  During  this 
stage  only  a  strongly  positive  reaction  should  be  re- 
garded as  significant.  In  late  and  especially  in  latent 
syphilis  the  reaction  again  grows  weaker.  More  sig- 
nificance may,  therefore,  attach  to  weak  reactions  in 
such  cases. 

4.  A  ^  positive  reaction  quickly  becomes  negative 
under  specific  treatment,  to  recur  if  treatment  is  inade- 
quate. Apparently  cured  cases  may  show  a  positive 
reaction  six  months  to  a  year  after  a  "provocative"  dose 
of  salvarsan. 

5.  The  behavior  of  the  blood  is  no  guide  as  to  the 


COMPLEMENT   DEVIATION    TEST  63 1 

condition  of  the  central  nervous  system.  Recent  inves- 
tigations have  shown  that  the  central  nervous  system 
becomes  involved  very  early  in  practically  all  cases, 
and  the  organisms  so  located  are  peculiarly  inaccessible 
to  attack  by  present  methods,  .  No  case  may  be  re- 
garded as  cured  until  both  blood  and  cerebrospinal 
fluid  show  a  persistent  normal  condition. 

Routine  Methods. — The  labor  involved  in  carrying  out 
the  somewhat  elaborate  details  of  the  method  as  above  out- 
lined may  be  materially  lightened  by  systematizing  the 
work  somewhat  as  follows:  On  the  day  before  the  tests  are 
to  be  made  prepare  an  abundance  of  clean  glassware  and  the 
red  cell  emulsion;  see  that  the  water-bath  is  properly  reg- 
ulated; and,  if  desired,  bleed  one  or  more  guinea-pigs  for 
complement,  and  place  the  blood  in  the  ice-box.  In  the 
morning  proceed  as  follows: 

1.  Set  up  the  complement  titration,  place  in  the  incuba- 
tor, and  mark  the  time. 

2.  Pipet  ofiF  the  sera  to  be  tested  into  clean  test-tubes, 
and  place  in  the  water-bath  to  inactivate.     Mark  the  time. 

3.  Arrange  in  the  rack  the  tubes  needed  for  the  test,  the 
antigen  control,  and  the  amboceptor  titration. 

4.  By  the  time  inactivation  is  complete  the  complement 
titration  will  be  nearly  or  quite  finished.  Forty-five 
minutes  will  suffice  for  the  latter.  Now  set  up  the  tests 
proper,  with  the  controls  and  the  amboceptor  titration. 

5.  At  the  end  of  the  hour  the  titration  may  be  read,  and 
the  indicated  amount,  and  the  red  cells,  added  to  all  tubes. 
Two  hours  later  the  final  result  is  read  and  recorded. 
Glassware  should  immediately  be  washed  and  put  away  for 
the  next  occasion.  A  little  experience  will  enable  one  to 
make  from  twenty-five  to  fifty  tests  between  9  a.m.  and 
3  P-m. 


632  SERODIAGNOSTIC   METHODS 

B.  Complement  Deviation  Test  for  Gonorrhea 
Method  of  Schwartz  and  McNeil 

The  method  as  given  below  represents  minor  modifi- 
cations of  the  original  method  suggested  by  experience 
in  the  writer's  laboratory.  The  antigen  is  an  autolysate 
of  a  large  number  of  strains  of  the  gonococci.  It  may  be 
obtained  from  Parke,  Davis  &  Co.  For  use,  dilute  with 
9  parts  of  salt  solution.  The  amount  used  for  the  test 
is  one  which  gives  a  strong  positive  reaction  with  a 
known  positive  serum  or  with  the  antigonococcic  serum 
of  Torrey  (also  marketed  by  Parke,  Davis  &  Co.), 
provided  this  amount  is  not  anticomplementary.  In 
our  experience  o.  1 5' c.c.  of  a  10  per  cent,  dilution  has  met 
these  conditions 

The  complement  is  used  in  a  10  per  cent,  dilution. 
Complement  and  amboceptor  are  titrated  against  o.i 
c.c.  of  5  per  cent,  sheep  cell  emulsion,  instead  of  i  c.c. 
The  same  quantity  of  red  cell  emulsion  is,  of  course, 
also  used  in  making  the  test. 

The  patient's  serum  is  used  inactivated.  In  the  orig- 
inal method  the  test  is  carried  out  with  0.05,  o.io,  and 
0.15  c.c.  In  our  experience  0.05  c.c.  is  almost  invari- 
ably negative,  while  0.15  c.c.  is  almost  invariably  anti- 
complementary.    We  have,  therefore,  used  only  o. i  c.c.  ^ 

In  other  respects  the  test  is  carried  out  exactly  like 
the  test  for  syphilis.  The  reaction  is  negative  during 
the  acute  stages  of  the  disease,  but  is  useful  in  deter- 
mining the  presence  of  a  focus  of  chronic  infection.  Its 
chief  importance  lies  in  the  fact  that  it  becomes  negative 

^  My  assistant,  Dr.  T.  F.  Walker,  has  kindly  furnished  me  with 
these  data,  based  on  an  unusually  extensive  experience  in  my 
laboratory  with  the  method. 


COMPLEMENT   DEVIATION    TEST  633 

in  a  short  time  (probably  about  two  weeks)  after  a  cure 
is  completed. 

C.  Complement  Deviation  Test  for  Tuberculosis 
Method  of  Hammer 

The  antigen  is  a  mixture  of  Koch's  old  tuberculin 
and  an  extract  of  tuberculous  granulation  tissue  freed 
as  much  as  possible  from  other  tissue.  Tissues  from  a 
surgical  lesion,  such  as  the  knee,  are  most  suitable. 
Cover  the  tissue  witti  4  parts  of  alcohol  and  extract  for 
three  to  five  days.  Filter,  and  dilute  the  filtrate  with 
3  parts  of  salt  solution  for  use.  Test  0.4,  0.2,  and 
0.1  c.c.  of  this  against  o.i  c.c.  of  known  positive  serum. 
Or,  cover  the  tissue  w^ith  9  parts  of  acetone  and  extract 
for  ten  days.  Filter,  and  evaporate  to  dryness  at  37°C. 
Take  up  the  residue  in  an  equal  volume  of  alcohol  and 
dilute  for  use  with  10  volumes  of  salt  solution.  Titrate 
as  above.  In  either  case  the  dose  used  is  the  largest, 
twice  which  is  not  anticomplementary. 

Now  add  to  9  volumes  of  the  diluted  extract  i  volume 
of  old  tuberculin,  and  repeat  the  titration  as  above. 
The  dose  is  determined  according  to  the  same  rule. 
A  certain  proportion  of  cases  will  react  with  one  or 
other  of  the  antigens  alone,  but  the  larger  percentage 
of  positive  results  will  be  obtained  with  the  mixed 
antigen. 

Arrange  the  tubes  as  for  the  Wassermann  reaction. 
In  all  the  tubes  place  i  c.c.  of  5  per  cent,  complement 
serum.  To  the  front  tubes  add  the  titrated  dose  of 
antigen.  In  each  pair  of  tubes,  front  and  rear,  place 
0.1  c.c.  of  the  several  sera  respectively,  inactivated  at 
56°C.  for  thirty  minutes.     Bring  all  the  tubes  to  a  like 


634  SERODIAGNOSTIC   METHODS 

volume,  mix,  and  let  stand  for  three  hours,  at  room-tem- 
perature. Add  2  units  of  amboceptor  and  i  c.c.  of  5 
per  cent,  red  cell  emulsion.  Mix,  and  place  in  the  in- 
cubator for  one  hour.  The  tests  are  then  ready  for  the 
final  reading. 

Method  of  Craig 

Craig  has  recently  proposed  a  modification  which 
gives  in  his  hands  a  smaller  proportion  of  positive 
results  in  non-tuberculous  cases.  The  antigen  is 
made  by  growing  several  strains  of  the  human  bacillus 
on  an  alkaline  bouillon  containing  a  teaspoonful  of 
aseptically  removed  egg-white  and  egg-yolk  for  each 
250  c.c.  of  bouillon.  When  growth  is  well  advanced 
add  an  equal  amount  of  95  per  cent,  alcohol,  and 
shake  in  a  shaking  machine  for  twelve  hours;  then 
place  in  the  incubator  for  twenty-four  hours,  shake 
again  for  six  hours,  and  finally  filter  through  a  very 
fine  filter-paper  or  a  Berkefeld  filter.  The  mixed 
filtrates  constitute  the  antigen,  which  is  usually 
used  without  diluting.  The  antigen  must  be  kept 
constantly  in  the  ice-box  and  amounts  needed  for  mak- 
ing tests  removed  aseptically  as  required.  It  is 
titrated,  without  diluting,  for  antigenic,  anticomple- 
mentary, and  hemolytic  properties  as  given  under  the 
Wassermann  reaction.  One  antigenic  unit  is  used 
for  the  test.  The  serum  to  be  tested  is  collected  as 
for  the  Wassermann  reaction,  and  inactivated  at  56° 
for  thirty  minutes.  Four  capillary  drops  are  used  for 
the  test.  The  complement  used  is  fresh  guinea-pig 
serum  diluted  with  1)4  parts  salt  solution,  and  titrated 
against  o.i  c.c.  of  10  per  cent,  washed  human  red  cells, 
of  which  2  units  (usually  about  o.i  c.c.)  are  used.     The 


COMPLEMENT    DEVIATION    TEST  635 

antihuman  amboceptor  may  be  purchased,  or  may  be 
prepared  by  injecting  each  of  several  well-grown,  healthy 
rabbits  with  three  doses  of  thoroughly  washed  human 
red  cells,  made  up  after  the  final  centrifugation  with 
salt  solution  to  about  one-half  the  original  volume  of  the 
blood.  The  first  dose  is  5  c.c.  given  under  the  skin  of 
the  abdomen;  the  second  and  third  doses  are  of  3  c.c. 
given  in  the  marginal  vein  of  the  ear.  These  doses 
are  given  at  intervals  of  five  or  six  days,  and  nine  days 
after  the  last  dose  a  small  amount  of  blood  is  drawn 
from  the  ear  of  each  rabbit  and  titrated.  If  one  or 
two  capillary  drops  of  a  1-40  dilution  completely  hemo- 
lyzes  0.1  c.c.  of  10  per  cent,  suspension  of  washed 
human  red  cells,  in  one  hour  in  the  incubator,  in  the 
presence  of  i  unit  of  complement,  the  amboceptor  is 
strong  enough,  and  the  rabbit  should  be  killed  while  in 
a  fasting  condition,  by  cutting  the  carotid  artery.  Col- 
lect the  blood  in  a  clean  dish,  and  after  the  serum  has 
separated,  preserve,  preferably  by  impregnating  a 
suitable  filter-paper  with  it.  Craig  uses-  Schleicher 
and  Schilll,  No.  597. 

The  following  summarizes  the  differences  in  the 
technic  of  the  test  and  that  of  the  Wassermann  re- 
action already  described: 

(a)  Four  capillary  drops  of  the  (inactivated)  serum 
to  be  tested,  instead  of  0.2' c.c. 

(b)  0.1  c.c.  of  10  per  cent.,  or  i.o  c.c.  of  i  per  cent, 
washed  human  red  cells,  instead  of  i.o  c.c.  of  5  per  cent, 
washed  sheep  cells. 

(c)  Antihuman,  instead  of  antisheep  amboceptor. 

(d)  Two  units  of  40  per  cent,  guinea-pig  serum,  as 
titrated  against  {b)  and  (c)  (usually  o.i  c.c). 


636  SERODIAGNOSTIC    METHODS 

(e)  One  antigenic  unit  of  the  above  described  antigen. 
The  stock  antigen  is  usually  used  undiluted. 

V.  COBRA- VENOM  TEST  FOR  SYPHILIS 

Of  the  several  cobra-venom  reactions,  the  method  of 
Weil,  for  the  diagnosis  of  syphilis,  possesses  the  greatest 
practical  value,  and  is  here  given.  It  appears  to  depend 
upon  the  same  disturbance  of  lipoid  metaboKsm  which  is 
responsible  for  the  Wassermann  reaction.  It  is  known 
that  syphilis  is  characterized  by  a  withdrawal  of  lipoids 
from  their  chief  depots,  viz. :  the  central  nervous  system 
and  the  red  blood-cells,  with  a  marked  increase  of  the 
same  in  the  fluid  part  of  the  blood.  Since  it  is  also 
known  that  the  hemolytic  action  of  the  cobra  venom 
depends  upon  its  activation  by  lecithin,  in  other  words, 
upon  a  lecithin-venom  complex  in  which  the  lecithin 
serves  as  complement,  it  may  fairly  be  assumed  that 
the  loss  of  lipoids  by  the  red  cells  is  responsible  for  the 
increased  resistance  to  hemolysis  by  cobra  venom  upon 
which  Weil's  reaction  is  based. 

1.  Materials  Required. — i.  The  cobra  venom  may  be 
obtained  from  Poulenc  Freres,  Paris.  Weil's  stock 
solution  is  a  0.5  per  cent,  solution  in  0.9  per  cent,  salt 
solution,  made,  of  course,  very  accurately.  It  deterio- 
rates very  rapidly  unless  kept  frozen.  For  this  reason 
I  have  used  very  successfully  the  solvent  usually  em- 
ployed for  the  purpose  of  other  reactions  in  this  group, 
viz.,  a  I  per  cent,  solution  of  venom  in  equal  parts  of 
distilled  water  and  chemically  pure  glycerin.  Before  it 
is  used,  this  should  be  allowed  to  stand  several  days  in 
the  ice-box,  where  it  keeps  extraordinarily  well. 

2.  The  blood-cells  to  be  tested.     Have  ready  normal 


COBRA-VENOM    TEST   FOR    SYPHILIS  637 

salt  solution  to  which  2  per  cent,  sodium  citrate  is 
freshly  added,  and  which  has  been  cooled  in  the  ice-box. 
Into  about  10  c.c.  of  this,  contained  in  a  graduated 
centrifuge  tube,  discharge  about  2  c.c.  of  the  patient's 
blood.  Do  not  shake.  The  red  cells  must  stand  in  con- 
tact with  the  citrate  solution  over  night  in  the  ice  box 
before  proceeding  to  the  test.  Wash  at  least  four  times 
with  0.9  per  cent,  salt  solution.  The  last  washing  of  all 
bloods  in  a  series  is  done  at  the  same  speed  and  for  the 
same  length  of  time.  Accurate  and  uniform  dilution  of 
the  cells  is,  of  course,  an  absolute  essential  to  obtain 
comparable  readings.  Pipet  off  the  last  wash-water 
and  make  up  to  a  4  per  cent,  emulsion  by  adding  24  vol- 
umes of  solution  to  the  i  volume  of  cells  as  read  in  the 
graded  tube  before  they  are  disturbed  The  salt  solu- 
tion used  for  washing  and  diluting  should  be  ice  cold 
and  the  final  emulsion  should  be  placed  on  ice  several 
hours  before  the  test  is  made. 

2.  Method. — ^From  the  stock  solution  of  venom  pre- 
pare the  following  solutions  for  the  test:  1-10,000, 
1-20,000,  1-30,000,  1-40,000.  Arrange  a  suitable  rack 
with  4  tubes  for  each  test.  In  the  respective  tubes  of 
each  row  place  i  c.c.  of  the  several  venom  solutions  and 
I  c.c.  of  the  cell  emulsion.  Incubate  for  one  hour  at 
37°C.  Mix  thoroughly  by  gentle  shaking  and  place  in 
the  ice-box  over  night.  In  the  morning  again  mix  thor- 
oughly and  make  the  final  reading  an  hour  later.  The 
result  will  depend  on  comparison  with  known  normal 
cells.     Something  like  the  following  may  be  anticipated : 

No  hemolysis  at  i-io,ooo  =  strongly  positive. 

Moderate  hemolysis  at  1-20,000  =  positive. 

Partial  hemolysis  at  1-30,000  =  negative. 

Complete  hemolysis  at  1-40,000  =  hypersensitive. 


638  SERODIAGNOSTIC   METHODS 

The  test  appears  later  in  the  disease  than  the  Wasser- 
mann  reaction,  and  yields  a  higher  percentage  of 
positive  results  in  late  latent  syphilis.  Furthermore,  it 
yields  less  quickly  to  treatment.  It  is  unquestionably 
an  important  aid  to  diagnosis  and  treatment  in  the 
class  of  cases  indicated. 


APPENDIX 

I.  STAINING  SOLUTIONS 

In  this  section  are  given  the  formulae  for  staining 
fluids  which  have  general  use,  particularly  for  identifica- 
tion of  bacteria.  Blood-stains  and  others  which  are 
used  only  for  special  purposes  are  discussed  in  the  body 
of  the  book  and  may  be  found  by  consulting  the  Index. 

1.  Carbol  Thionin. — Saturated  solution  thionin  in 
50  per  cent,  alcohol,  20  c.c;  2  per  cent,  aqueous  solution 
phenol,  100  c.c. 

This  stain  is  especially  useful  in  counting  bacteria 
for  standardization  of  vaccines  (see  p.  590).  It  can  be 
used  as  a  general  stain.  In  blood  work  it  is  used  for  the 
malarial  parasite  and  for  demonstration  of  basophilic 
degeneration  of  the  red  cells.  The  fluid  is  applied  for 
one-half  to  three  minutes,  after  fixation  by  heat,  or 
about  a  minute  in  saturated  aqueous  solution  of 
mercuric  chlorid  or  i  per  cent,  formalin  in  alcohol. 

2.  Fuchsin. — This  dye  should  not  be  confused  with 
acid  fuchsin.  Its  solutions  are  generally  made  with 
phenol  as  a  mordant  and  they  are  then  very  powerful 
bacterial  stains,  with  a  strong  tendency  to  over-staining. 
They  are  used  chiefly  for  the  tubercle  bacillus. 

Czaplewski's  carbol-fuchsin  is  superior  to  the  widely 
used  Ziehl  solution  in  that  it  acts  more  quickly  and  is 
permanent.    To  i   Gm.  fuchsin  and  5  c.c.  liquefied 

639 


640  APPENDIX 

phenol,    add    50  c.c.  glycerol  with  constant  stirring; 
and  finally  add  50  c.c.  water,  mix  well,  and  filter. 

3.  Gentian  Violet. — The  combinations  given  below 
are  powerful  bacterial  stains  which  have  their  chief 
use  in  Gram's  method.  They  may  be  used  interchange- 
ably, but  the  solution  with  phenol  is  probably  most 
serviceable.  Formalin-gentian-violet  remains  good  for 
years  but  is  less  satisfactory  for  Gram's  method  than 
the  others.  Methyl  violet  may  he  substituted  for  gentian 
violet  in  these  formulce,  and  is  preferable. 

Anilin-gentian  violet. — Ehrlich's  formula  is  the  one 
generally  used,  but  this  keeps  only  a  few  weeks. 
Stirling's  solution,  which  keeps  much  better  and  seems 
to  give  equal  results,  is  as  follows :  gentian  violet,  5  Gm. ; 
alcohol,  10  c.c;  anilin  oil,  2  c.c;  water,  88  cc 

Czaplewski's  Carbol-gentian-violet. — To  i  Gm. 
gentian  violet  and  5  c.c  liquefied  phenol,  add  50  c.c 
glycerol  with  constant  stirring;  finally  add  50  c.c 
water,  mix  well,  and  filter. 

Formalin-gentian-violet  consists  of  5  per  cent, 
solution  formalin,  75  parts;  saturated  alcoholic  solution 
gentian  violet,  25  parts. 

4.  Hematoxylin  is  one  of  the  best  nuclear  stains 
available.  There  are  many  combinations,  most  of 
which  require  weeks  or  months  for  "ripening."  The 
following  is  a  good  solution  which  is  ready  for  use  as  soon 
as  made. 

Harris'  Hematoxylin. — Dissolve  i  Gm.  hematoxylin, 
crystals  in  10  c.c  alcohol.     Dissolve  20  Gm.  ammonia 
alum  in  200  c.c.  distilled  water  with  the  aid  of  heat  and 
add   the   alcoholic   hematoxylin   solution.     Bring    the 
mixture  to  a  boil  and  add  half  a  gram  of  mercuric  oxide. 


STAINING    SOLUTIONS  64 1 

As  soon  as  the  solution  assumes  a  dark  purple  color, 
remove  the  vessel  from  the  flame  and  cool  quickly  in 
a  basin  of  cold  water. 

5.  lodin  is  used  as  a  part  of  Gram's  method  and  as 
a  special  stain  for  various  purposes.  For  starch,  a  very 
weak  solution  is  desirable;  for  Leptothrix  buccalis,  a 
strong  solution  such  as  Lugol's.  The  solutions  de- 
teriorate upon  long  standing. 

Gram's  lodin  Solution. — lodin,  i  Gm.;  potassium 
iodid,  2  Gm.;  water,  300  c.c. 

Lugol's  solution  {Liquor  lodi  Compositus,  U.  S.  P.) 
consists  of  iodin,  5  Gm.;  potassium  iodid,  10  Gm.; 
water,  100  c.c.  Gram's  iodin  solution  may  be  made 
from  this  by  adding  fourteen  times  its  volume  of  water. 

6.  Methylene -blue  is  a  widely  used  basic  dye  which 
does  not  readily  over-stain.  The  following  solutions 
are  useful: 

Gabbet's  Stain. — This  is  used  in  Gabbet's  method 
for  the  tubercle  bacillus.  It  consists  of  methylene- 
blue,  2  Gm.;  water,  75  c.c;  concentrated  sulphuric  acid, 
25  c.c. 

Loeffler's  alkaline  methylene -blue  is  one  of  the  most 
useful  bacterial  stains  for  general  purposes.  The 
solution  is  applied  at  room  temperature  for  30  seconds 
to  three  minutes,  and  is  followed  by  rinsing  in  water. 
Fixation  may  be  by  heat  or  chemicals.  The  stain  is 
composed  of  30  parts  of  a  saturated  alcoholic  solution 
<of  methylene-blue  and  100  parts  of  a  1:10,000  aqueous 
solution  of  caustic  potash.     It  keeps  indefinitely. 

Pappenheim's  methylene-blue  solution  is  used  as 
decolorizer  and  contrast  stain  in  Pappenheim's  method 
for   the   tubercle   bacillus.     Dissolve    i    Gm.    corallin 


642  APPENDIX 

(rosolic  acid)  in  100  c.c.  absolute  alcohol;  saturate  with 
methylene-blue ;  and  add  20  c.c.  glycerol. 

7.  Pyronin. — Used  in  strong  aqueous  solution,  this 
is  useful  as  a  contrast  stain  in  Gram's  method,  but 
results  are  more  satisfactory  when  the  dye  is  combined 
with  methyl  green. 

Pappenheim's  Pyronin-methyl-green  Stain. — This 
solution  colors  bacteria  red  and  nuclei  of  cells  blue. 
It  is,  therefore,  especially  useful  for  intracellular 
bacteria  like  the  gonococcus  and  the  influenza  bacillus. 
It  is  a  good  stain  for  routine  purposes,  is  a  most  excellent 
contrast  stain  for  Gram's  method,  and  is  also  used  to 
demonstrate  Dohle's  inclusion  bodies  in  the  blood. 
It  colors  the  cytoplasm  of  lymphocytes  bright  red,  and 
has  been  used  as  a  differential  stain  for  these  cells. 
The  solution  is  applied  cold  for  one-half  to  five  minutes. 
It  consists  of  saturated  aqueous  solution  methyl-green, 
3  to  4  parts,  and  saturated  aqueous  solution  pyronin, 
I  to  13=-^  parts.  It  is  a  good  plan  to  keep  these  solutions 
in  stock  and  to  mix  a  new  lot  of  the  staining  fluid  about 
once  a  month.  If  it  stains  too  deeply  with  either  dye, 
the  proper  balance  is  attained  by  adding  a  little  of  the 
other. 

8.  Simple  Bacterial  Stains. — A  simple  solution  of 
any  basic  anilin  dye  (methylene-blue,  basic  fuchsin, 
gentian-violet,  etc.)  will  stain  nearly  all  bacteria. 
These  simple  solutions  are  not  much  used  in  the  clinical 
laboratory,  because  other  stains,  such  as  Loffler's 
methylene-blue  and  Pappenheim'i  pyronin-methyl- 
green  stain,  which  serves  the  purpose  even  better,  are 
at  hand. 


OFFICE    LABORATORY   EQUIPMENT  643 

9.  Sudan  m  is  a  valuable  stain  for  fat,  to  which  it 
gives  an  orange  color.  Scharlach  R  is  a  similar  but 
stronger  dye,  and  may  be  substituted  to  advantage. 
They  may  be  used  as  a  saturated  solution  in  70  per  cent, 
alcohol  or  in  the  following  combination. 

Herxheimer's  Sudan  IH  consists  of  equal  parts  of 
70  per  cent,  alcohol  and  acetone  saturated  with  Sudan 
III  (or  Scharlach  R). 

n.  OFFICE  LABORATORY  EQUIPMENT 

It  is  not  to  be  expected  that  a  physician  in  active 
practice  will  make  routine  use  of  all  the  methods 
described  in  this  book.  Although  he  will  need  nearly 
all  of  them  for  the  study  of  his  more  difficult  cases,  his 
daily  laboratory  work  will  probably  be  limited  to  a 
few  simple  procedures.  With  this  in  mind  the  follow- 
ing list  of  laboratory  procedures  is  suggested  as  the 
minimum  with  which  a  physician  should  be  thoroughly 
familiar  and  upon  which  he  may  build  as  his  practice 
requires.  The  methods  are  selected  because  of  their 
simplicity  and  practical  usefulness. 

METHODS  FOR  OFFICE  ROUTINE 

Sputum 

Careful  inspection  (see  p.  59). 

Simple  microscopic  examination  unstained  (see  p.  63). 

Examination  for  tubercle  bacilli  (see  p.  76). 

Urine 
Reaction  (see  p.  106). 
Specific  gravity  (see  p.  107). 
Calculation  of  total  solids  (see  p.  no). 
Phenolsulphonephthalein  test  of  kidney  function  (see  p.  112). 


644  APPENDIX 

Albumin,  qualitative: 

Roberts's  ring  test  (see  p.  154). 

Purdy's  heat  test  (see  p.  156). 
Albumin,  quantitative: 

Esbach's  test  (seep.  157). 
Sugar,  qualitative: 

Benedict's  test  (see  p.  163). 
Sugar,  quantitative: 

Benedict's    method    (see    p.    167),    or    Roberts's    yeast    method 
(see  p.  170). 
Acetone,  Lange's  or  Rothera's  test  (see  p.  176). 
Diacetic  acid,  Gerhardt's  test  (see  p.  177). 
Bile,  Gmelin's  test  (see  p.  180). 
Hemoglobin,  benzidin  test  (see  p.  182). 
Indican,  Obermayer's  test  (see  p.  134). 
Microscopic  examination  (see  p.  198). 

Blood 

Coagulation  time,  simple  method  (see  p.  258). 
Hemoglobin,  Dare  or  Sahli  method  (see  pp.  266  to  268). 
Red  corpuscle  count  (see  p.  272). 
Color  index  calculation  (see  p.  284). 
Leukocyte  count  (see  p.  294). 
Differential  leukocyte  count  (see  p.  324). 

Microscopic  examination  of  stained  films  for  pathological  red  cells 
and  malarial  parasites  (see  pp.  315,  357). 

Stomach  Contents 

Careful  inspection  (see  p.  398). 

Total  acidity,  Topfer's  method  (see  p.  408). 

Free  hydrochloric  acid,  Topfer's  method  (see  p.  410). 

Lactic  acid,  Kelling's  test  (see  p.  403). 

Microscopic  examination  (see  p.  414). 

Feces 

Careful  inspection  (see  p.  424). 

Occult  blood,  benzidin  test  after  extraction  with  ether  (see  p.  429). 

Microscopic  examination: 

(a)  for  parasites  or  their  ova  (see  p.  443). 

(b)  to  ascertain  state  of  digestion  (see  pp.  437,  444). 


OFFICE    LABORATORY    EQUIPMENT  645 

Serum  Methods 

Widal  test  by  macroscopic  method,  using  one  of  the  commercial 
outfits  (see  p.  608). 

Miscellaneous 

Microscopic  examination  of  pus: 

(a)  simple  stain  (see  p.  571). 

(b)  Gram's  method  (see  p.  572). 
Puncture  fluids: 

(a)  Careful  inspection  (see  pp.  520  and  524). 
(6)    Microscopic  examination  for    bacteria     and  differential  cell 
count  (see  p.  521). 

Syphilitic    material    for    spirochetes,    Giemsa    stain    or    India-ink 
method  (see  pp.  550-SS3)- 
Milk: 

Fat,  Leffmann-Beam  method  (see  p.  546). 
Protein,  by  calculation  (see  p.  545). 

A  list  of  equipment  which  is  sufficient  for  all  the 
above-mentioned  methods  (and  for  many  others  in 
addition)  is  given  below.  The  total  cost,  exclusive  of 
the  furniture,  but  including  a  first  class  microscope, 
simple  mechanical  stage,  and  Sahli  hemoglobinometer 
will  be  about  $130.00  in  normal  times.  ^  There  is  no  real 
economy  in  purchasing  instruments  of  inferior  quality. 

A.  FURNITURE 

A  table,  with  drawer,  and  a  few  shelves  for  bottles 
and  glassware  constitute  the  only  really  essential 
laboratory  furniture  even  for  fairly  extensive  work. 
When  a  special  room  is  not  available  these  may  stand 
behind  a  screen  in  the  physician's  consulting  room. 
Gas  and  running  water  are  very  desirable,  but  not 
absolutely  necessary. 

^  The  entire  outfit,  with  ready-prepared  reagents  and  staining  solu- 
tions, can  be  purchased  of  Paul  Weiss,  1620  Arapahoe  St.,  Denver; 
A.  H.  Thomas  Co.,  West  Washington  Square,  Philadelphia,  and  proba- 
bly many  other  supply  houses. 


646  APPENDIX 

The  shelves  may  conveniently  take  the  form  of  a 
shallow  case  without  doors,  which  stands  upon  the  back 
of  the  table  and  which,  in  addition  to  the  shelves,  has 
two  tall  compartments,  one  for  the  combined  buret  and 
filter  stand,  the  other  for  the  microscope  in  its  case.  If 
space  allows,  however,  it  will  be  found  more  satisfactory 
to  keep  the  microscope  under  a  glass  bell-jar  (or  paste- 
board cover,  p.  41)  on  a  stand  or  small  table  before  a 
window.  It  is  thus  always  ready  for  use  and  is  away 
from  the  neighborhood  of  corroding  chemicals.  A  stool 
or  chair  of  the  proper  height  (see  p.  37)  should  be 
at  hand. 

A  convenient  reservoir  for  wash  water  is  a  large 
bottle,  which  stands  up)on  the  top  shelf  and  from 
which  water  is  siphoned  by  means  of  a  rubber  tube 
with  a  medicine-dropper  tip  and  a  Mohr  pinch  cock. 
The  glass  tip  should  hang  directly  over  a  miniature  sink 
consisting  of  a  large  glass  funnel  whose  stem  passes 
through  the  table  top  and  drains  by  means  of  a  rubber 
tube  into  an  earthen  jar  below.  All  staining  should  be 
done  over  this  fuimel-sink,  the  slides  being  supported 
upon  a  rack  consisting  essentially  of  two  small  rods 
about  2  inches  apart  placed  across  the  top  of  the  funnel. 

The  following  wood  finish  is  extensively  used  for  table 
tops  in  the  laboratories  of  this  country'.  It  gives  an 
ebony-black  surface  which  resists  practically  all  reagents. 
The  wood  must  be  new,  or  at  least  not  painted,  varnished, 
or  waxed. 

Solution  No.  i. 

Copper  sulphate  125  Gm. ; 

Potassium  chlorate  (or  permanganate)  125  Gm.; 

Water  1000  c.c. 


OFFICE    LABORATORY   EQUIPMENT  647 

Solution  No.  2. 

Anilin  oil  120  c.c; 

Hydrochloric  acid,  concentrated  180  c.c; 

Water  1000  c.c. 

Apply  two  coats  of  Solution  No.  i,  hot,  and  then  two 
coats  of  Solution  No.  2,  without  heating,  allowing  each 
coat  to  dry  thoroughly  before  the  next  is  applied.  When 
the  last  coat  is  dry  remove  the  excess  of  the  chemicals  by 
rubbing  with  a  coarse  cloth.  Finally  rub  thoroughly  with 
a  mixture  of  equal  parts  of  turpentine  and  linseed  oil. 

B.  APPARATUS 

1  Basin  of  white  enameled  ware. 

2  Beakers  with  lip,  about  50  c.c.  capacity.  Small  coflFee 
cups  may  be  substituted. 

I  Blood-lancet  or  some  substitute,  as  a  Hagedorn  needle 
(see  p.  253). 

I  Bunsen  burner  with  rubber  tubing,  the  small  "micro" 
burner  being  especially  satisfactory.  An  alcohol  lamp  will 
answer. 

I  Buret,  25  c.c.  capacity,  preferably  with  Schellbach 
stripe.  An  accurate  10  c.c.  graduated  pipet  may  be  used 
for  much  work  but  is  not  so  satisfactory  as  the  buret. 

I  Centrifuge,  hand,  electric  or  water  power  (see  Figs.  ^^, 
34).  The  last  is  cheap  and  satisfactory.  Metal  shields 
with  flat  bottoms  and  rubber  cushions  are  preferable  to 
the  ordinary  conical  aluminum  shields  because  they  allow 
the  use  of  ordinary  test-tubes  as  well  as  conical  centrifuge 
tubes. 

I  Esbach  tube  (see  Fig.  42). 

1  Pack  filter  papers,  round,  about  12  cm.  in  diameter, 
good  quality. 

I  Funnel,  glass,  about  7  cm.  in  diameter. 


648  APPENDIX 

4  feet  Glass  tubing,  about  7  or  8  mm.  outside  di.ameter, 
of  soft  glass  for  making  urine  pipets,  etc. 

I  Graduate,  cylinder,  100  c.c,  double  graduations.  This 
is  used  chiefly  for  making  solutions. 

I  Hemacytometer  (see  Figs.  98, 102,  103).  Probably  the 
most  satisfactory  outfit  consists  of  a  Buerker-type  counting 
chamber  with  Neubauer  ruling,  a  "red  pipet"  and  a  "white 
pipet." 

I  Hemoglobinometer.  The  Dare  will  probably  be  found 
most  convenient  if  the  price  is  not  prohibitive;  otherwise 
the  Sahli  (or  Kuttner)  is  recommended.  A  Tallquist  book 
should  be  carried  in  the  hand-bag. 

i  Pack  lens-cleaning  paper.  Two  rows  of  stitching, 
3^^  inch  apart,  may  be  run  across  the  middle  of  the  package 
on  the  sewing  machine  and  the  package  then  cut  into 
little  booklets  of  convenient  size. 

I  Box  labels  for  bottles.  Denison's  No.  A-4  is  a  useful 
size. 

I  Box  labels  for  slides. 

I  Mechanical  stage,  attachable  (see  p.  49). 

4  Medicine  droppers:  two,  labeled  "Stain"  and  "Water" 
respectively,  to  be  reserved  for  use  with  Wright's  blood- 
stain; one,  which  delivers  the  proper  sized  drop,  to  be 
reserved  for  the  quantitative  sugar  estimation. 

I  Eye-piece  micrometer.  The  card-board  micrometer  made 
as  described  on  p.  44  will  answer  for  most  clinical  work. 

I  Microscope  equipped  as  described  on  p.  48. 

50  Micro  cover-glasses,  No.  2  thickness.  The  22  mm. 
squares  are  most  convenient  for  general  purposes. 

I  Box  (3-^  gross)  micro  slides,  75  X  25  mm.,  clear  white 
glass,  medium  thickness,  ground  edges. 

I  Pencil,  wax,  for  writing  on  glass,  red  or  blue. 

I  Petri  dish  with  cover,  about  15  cm.  in  diameter. 

I  Pipet,  10  c.c,  graduated. 

I  Rule,  celluloid,  6  inches  and  15  cm.     These  are  sold  by 


OFFICE    LABORATORY    EQUIPMENT  649 

Bausch  and  Lomb  Optical  Co.  and  Spencer  Lens  Co.  for 
five  cents  each. 

I  Stand  for  filter,  buret,  etc.  Convenient,  but  not  abso- 
lutely essential. 

I  Stomach  tube.  The  Rehfuss  type  is  required  if  frac- 
tional method  of  examination  is  employed  and  is  best  for 
all  purposes. 

I  Test-glass,  conical    A  wine-glass  will  serve. 

12  Test-tubes,  size  about  125  X  16  mm.,  without  flange. 

I  Test-tube  brush,  bristle,  with  tuft  at  tip. 

I  Test-tube  rack  holding  six  tubes. 

I  Urinometer  with  cylinder.  Must  have  wide  gradua- 
tions.    Test  with  distilled  water. 

I  Widal  test  outfit,  macroscopic  method.  Satisfactory 
outfits  are  sold  under  various  trade  names,  ''Tvohoid 
agglutometer,"  etc. 

I  Box  wooden  toothpicks. 

C.  REAGENTS  AND  STAINS 

All  staining  solutions  and  many  reagents  are  best 
kept  in  small  dropping  bottles,  of  which  the  "flat- 
topped  T.  K."  pattern  is  most  satisfactory.  Other 
reagents  may  be  kept  in  ordinary  round  prescription 
bottles  of  4  to  8  ounces'  capacity.  Bottles  containing 
highly  volatile  reagents  should  be  sealed  with  paraffin 
if  not  in  constant  use;  while  those  containing  strong 
caustic  soda  solutions  should  have  rubber  stoppers. 

Most  staining  solutions  and  chemical  reagents 
can  be  purchased  ready  prepared.  For  the  physician 
who  does  only  a  small  amount  of  work  the  "Soloid" 
tablets  manufactured  by  Burroughs,  Welcome  &  Co. 
are  convenient  and  satisfactory.  These  tablets  have 
only   to   be   dissolved  in   a   specified   amount   of   the 


650  APPENDIX 

appropriate  fluid  to  produce  the  finished  solution. 
Most  of  the  stains  and  many  of  the  commoner  reagents 
are  supplied  in  this  form. 

If,  however,  his  time  permits  the  physician  will  find  it 
more  satisfactory  and  much  more  economical  to  prepare 
his  own  solutions,  with  exception  of  normal  solutions 
and  a  very  few  stains. 

Reagents 

50  c.c.  Acid,  acetic,  glacial,  99}^^  per  cent.  Other 
strengths  can  be  made  from  this  as  desired. 

50  c.c.  Acid,  hydrochloric,  C.P.,  Sp.  Gr.  i.  16.  Contains 
about  32  per  cent.  HCl.  An  approximate  decinormal  solu- 
tion for  use  with  the  Sahli  hemoglobinometer  can  be  made 
by  adding  12  c.c.  of  this  acid  to  988  c.c.  distilled  water. 

50  c.c.  Acid,  nitric,  C.P.  Yellow  nitric  acid  can  be  made 
from  this  by  adding  a  splinter  of  pine  (match  stick)  or 
allowing  it  to  stand  in  the  sunlight  for  a  short  time. 

50  c.c.  Acid,  sulphuric,  C.P. 

50  c.c.  Alcohol,  amylic,  C.P.  Used  in  the  estimation  of 
fat  in  milk. 

200  c.c.  Alcohol,  ethylic  (grain  alcohol).  This  is  ordi- 
narily about  93  to  95  per  cent,  and  other  strengths  can  be 
made  as  desired.  Whenever  the  word  "alcohol"  is  used  in 
the  text  without  qualification,  this  alcohol  is  meant. 

100  c.c.  Alcohol,  methylic,  Merck's  "Reagent,"  for  making 
Wright's  blood  stain.  May  be  omitted  if  the  stain  is  pur- 
chased ready  prepared. 

100  c.c.  Ammonium  hydroxid  (strong  ammonia)  Sp.  Gr. 
0.9. 

2cp  C.C.  Benedict's  solution  for  qualitative  sugar  test 
(seep.  163). 

100  c.c.  Benedict's  solution  for  quantitative  sugar  estima- 
tion (see  p.  167). 


OFFICE   LABORATORY   EQUIPMENT  65 1 

10  Gm.  benzidin.     Specify  "for  blood  test." 

I  tube  Canada  balsam  in  xylol.  Necessary  only  if  per- 
manent mounts  are  to  be  made. 

100  c.c.  Chloroform,  U.  S.  P. 

100  c.c.  Diluting  fluid  for  red  corpuscle  count,  Hayem's 
preferred  (see  p.  279). 

100  c.c.  Diluting  fluid  for  leukocyte  count  (see  p.  300). 

30  c.c.  Dimethyl-amido-azo-benzol,  0.5  per  cent,  alcoholic 
solution. 

100  c.c.  Esbach's  solution  (see  p.  157). 

200  c.c.  Ether,  sulphuric,  U.  S.  P. 

30  c.c.  Ferric  chlorid,  10  per  cent,  aqueous  solution. 

100  c.c.  Formalin  (40  per  cent,  solution  of  formalde- 
hyd  gas).  The  expression  "10  per  cent,  formalin"  means 
I  part  of  this  40  per  cent,  solution  and  9  parts  of  water 
making  a  4  per  cent,  solution  of  formaldehyd  gas. 

50  c.c.  Hydrogen  peroxid,  U.  S.  P. 

I  Vial  litmus  paper,  Squibb,  red. 

1  Vial  litmus  paper,  Squibb,  blue. 

25  c.c.  Lugol's  solution  {Liquor  lodi  Composilus,  U.  S.  P.). 
Gram's  iodin  solution  (see  p.  641)  can  be  made  from  this  by 
adding  14  times  its  volume  of  water. 

50  Gm.  Magnesium  sulphate,  C.P.,  for  making  Roberts' 
solution  for  albumin  in  urine. 

100  c.c.  Obermayer's  reagent  for  indican  (see  p.  134). 

25  c.c.  Oil  of  cedar  for  immersion.  A  sufficient  quantity 
is  usually  supplied  with  the  microscope  when  purchased. 

25  c.c.  Phenolphthalein,  i  or  0.5  per  cent,  solution  in 
alcohol. 

2  Ampoules  phenolsulphonephthalein. 

50  Gm.  Sodium  chlorid,  C.P.,  for  Purdy's  albumm  test. 
Table  salt  may  be  used  but  is  not  so  good. 

1000  c.c.  Sodium  hydroxid,  decinormal  solution.  The 
practitioner  will  find  it  best  to  purchase  this  solution  ready 
prepared.     Most  chemical  supply  houses  carry  it  in  stock. 


652  APPENDIX 

For  rough  clinical  work  41  grams  of  Merck's  ''Sodium 
hydrate  by  alcohol"  from  a  freshly  opened  bottle  may  be 
dissolved  in  locxj  cc.  distilled  water.  This  makes  a  normal 
solution  and  must  be  diluted  with  9  volumes  of  water  to 
make  the  decinormal  solution. 

25  Gm.  Sodium  nitroprussid,  C.P.,  metals. 

50  Gm.  Talc,  purified  {Talcum  purijkalum,  U.  S.  P.)  or 
diatomaceous  earth  {Kiesdguhr)  for  clearing  urine. 

2000  cc  Water,  distUled.  In  some  regions  ordinary'  tjq>- 
water  answers  for  practically  all  purposes. 

Stains 

It  will  be  found  most  satisfactor\'  to  have  on  hand  a  stock 
of  dr\'  stains  (which  keep  well)  and  to  make  solutions  as 
needed.  Ordinarily  the  smallest  quantitj-  obtainable  in  an 
unbroken  package  should  be  purchased.  The  following 
dr>'  stains  make  up  a  fairly  complete  stock  for  the  clinical 
laboratorj^:  Fuchsin,  basic;  gentian  \*iolet;  methylene  blue, 
B,  X.;  methyl  green;  pyronin;  and  Wright's  stain.  Wright's 
stain  is  obtainable  in  i-Gm.  \-ials,  the  others  in  lo-Gm. 
vials.  The  most  frequently  used  solutions,  which  can  be 
purchased  in  25-cc  bottles,  are: 

Carbol-fuchsin  (see  p.  639). 

Carbol-gentian-^'iolet  (see  p.  640). 

Giemsa's  stain  (see  p.  313).  This  is  not  necessary  if  the 
India-ink  method  for  spirochetes  is  used. 

Loffler's  alkaline  methylene-blue  (see  p.  641). 

Pappenheim's  methylene-blue  contrast  stain  for  tubercle 
bacilli  (see  p.  641). 

Papp>enheim's  pyronin-methyl-green  stain  (see  p.  642). 

Wright's  blood  stain  (see  p.  309).  Much  of  the  solution 
on  the  market  is  unsatisfactory. 


^"EIGHTS   AND   MEASURES 


653 


m.  WEIGHTS,    MEASURES,  ETC,  WITH 
EQUIVALENTS 


Meter  (.mit  of  length; 


Cram  (onit  of  weigfct) : 
Liter  (onit  of  capacity) : 


METRIC 

\mimeter  (mm.)  —  ^^^ 
Cenrimeter  (cm.)  =  yl^  n 
KSooteter  =  xooo 

KHogram  (kilo.)    =  tooo  meters. 
Cubic  Centimeter  =  j^g  liter.       Same   mraanre  as 
liter  (ml.). 


Meter 


I  MflBmeter  =  (°-°3937  (A  *PPr°«-)  in. 
)  looo  microns. 

I  Centimeter  =  |°»37  (|^PP««->  *^ 
I  0.0328  feet, 
^f  39. 37  in. 
(  3  j8  feet. 

'^^        (0.001  milmneter. 


»S^  miw<  II  !•   =0.00135') 

a  Sq.  CcattBeter  =  0.1550    >sq.  m. 

« S4.  Ifecr  =  1550      ) 

I  S4-  Meter  =  10.76  sq.  feet. 


I  Inch         =  25.399  minimeters. 
I  Sq.  Inch  =  6.451  sq.  centimeters. 
1  Cb.  Inch  =  16.3S7  CQ.  centimeters. 


Gram 


f  '5-43  grains. 
I  0.563  dram 
0/335  omra 
=  i  ojooaa  pemi 

Io.357dBaik 
oMSpttmmu 
OjOOSKJ  pOBI 

tKiIognun  =  {S^ 
I  Liter  =  -<  6ijis  cs. 


1  Ca.  Cenfitiwtrr  =  ojofoo  /^^ 
I  Co.  Centimeter  ^  ojoox  fifer. 

1  Co.  Meter  =1^:?"-^ 

(^enus-fca.  m. 

I  Foot  =  30.48  centimeters. 

I  Sq.  Foot  =  0.093  sq.  meter. 
I  Co.  Foot  =  ou»8  en.  meter. 


AVOIRDUPOIS   WEIGHT 


I  GiaiB    =  OU165  {J^  approz.) 
s  Dtaat   =  1.77  (z{  approz.) 
I  C^mex  =  38.35  (3P*rfo^) 
I  Floaad  =  453-39  (y»apfiiia. 
I  Poaad  =  27.7  CO.  iachca. 
I  Poond  =  1. 215  lb.  Tmy. 


APOTHECARIES'   MEASURE 


I  Dram   =  60  minims. 
I  Oance  =  S  drams. 
I  Pint      =  16  ooaces. 
iGdOoa-^Si 


I  Dnun   =3.70 

I  ^ace  =  29.57 

I  Pte      =  473.1 

iGafcn  =  37«5^ 

s  GaBoo  =^x  OL  incarw. 


654 


APPENDIX 


APOTHECARIES'   WEIGHT 


X  Scruple  =  20  grains. 
1  Dram     =  3  scruples  =  60  gfrains. 
I  Ounce   =  8  drams  =  480  grains. 
I  Pound   =  12  ounces. 


=  0.065  ") 
=  3-887  I 

i  :=    ^I.IO     I 


I  Grain 

I  Dram 

I  Ounce  =  31 

I  Pound  =  373.2 


To  convert        minims 

"  "          jluidotinces 

"  "               grains 

"  "               drams 

"  "  cubic  cenlimelers 

"  "  cubic  cenlimelers 

"  "                 grams 

"  "               grams 


into  cubic  centimeters  multiply  by   0.061 
"    cubic  centimeters        "  29.57 

"  grams  "         "     0.0648 

"     3-887 


grams 

minims 

fluidounces 

grains 

drams 


16.23 

0.0338 
iS-432 

0.257 


TEMPERATURE 


Centigrade.         Fahrenheit. 

110° 230° 

100   212 

95   203 

90   194 

85   185 

80  176 

75   167 

70  158 

65  149 

60  140 

55   131 

50  122 

45   "3 

44   III. 2 

43   109-4 

42   107.6 

41   105-8 

40-5 104.9 

40  104 

39-5 103.1 

39   102.2 

385 101.3 

38  10Q.4 

37-5 99-5 


Centigrade. 


37° 

36-5 

36 

35-5 

35 

34 

33 

32 

31 

30 

25 

20 

15 
10 

+5 
o 

—5 
— 10 

—15 
— 20 


O.S4" 

I 

2 

2-5 


Fahrenheit. 

98.6° 

97-7 
96.8 

95-9 

95 

93-2 

91.4 

89.6 

87.8 

86 

77 
68 

59 

50 

41 

32 

23 

14 
+5 
—4 


I" 

1.8 

3-6 

4-5 


To  convert  Fahrenheit  into  Centigrade,  subtract  32 
and  multiply  by  0.555. 

To  convert  Centigrade  into  Fahrenheit,  multiply  by 
1.8  and  add  32. 


INDEX 


Absorption    spectra    of    hemo- 
globin derivatives,  370 
toxic,  degree  of,  331 
Absorptive  power  of  stomach,  419 
Accidental  albuminuria,  149 
Acetanilid  in  urine,  192 
Aceto-acetic  acid  in  urine,  177 
Acetone  bodies  in  urine,  172 
in   urine,    173.     See   also  Ace- 
tonuria. 
Acetonuria,  173 

detection  of  acetone  in,  174 
Frommer's  test  for,  177 
Gunning's  test  in,  175 
Legal's  test  for,  Lange's  modi- 
fications, 176 
Rothera's  test  for,  177 
Achlorhydria,  410 
Acholic  feces,  425 
Achromatic  objectives,  26 
Achromia,  316 
Achylia  gastrica,  gastric  contents 

in,  418 
Acid  fermentation  of  urine,  106 
hydrochloric,  combined,  392 

free,  393 
lactic,  in  gastric  contents,  401. 

See  Lactic  acid. 
urine,    unorganized    sediments 
in,  202,  203 
•Acid-fast  bacilli,  82 
Acidity,  quantitative  estimation 
of,  in  urine,  107 
Folin's  method,  107 
total,  of  gastric  contents,  408 
tests,  408 
Topfer's  test,  408 
Acidophilic  structures  of  blood, 

307 
Acids,  free,  in  gastric  contents, 
tests  for,  400 


Acids,  organic,  in  gastric  contents, 

401,  412 
Acquired  immunity,  600 
Actinomycosis  bovis  in  sputum, 

72 
Adulteration,     meat,     precipitin 

test  for,  614 
Agar-agar,  blood,  preparation  of, 
566 

glycerin,  preparation  of,  566 

preparation  of,  565 
Agglutination  of  bacteria,  607 

of  blood,  testing,  375 

of  erythrocytes,  376 
Agglutinins,  6oi 
Air-bubbles  in  urine,  241 
Albumin  in  sputum,  94 
Albuminometer,  Esbach's,  157 
Albuminuria,  149 

accidental,  149 

contact  tests  for,  152 

cyclic,  150 

detection,  152 

Esbach's   estimation   of   albu- 
min, 157 

estimation  of  albumin,  quanti- 
tative, 157 

false,  150 

from  blood  changes,  151 

from  kidney  changes,  151 

heat  and  nitric  acid  test,  156 

orthostatic,  150 

physiologic,  150 

postural,  150 

Purdy's     centrifugal    method, 
158 
heat  test,  156 
table     after    centrifugation, 

159 
renal,  150 
ring  tests  for,  152 


65s 


656 


INDEX 


Albuminuria,    Robert's   test   for, 

154 
sulphosalicylic  acid  test  for,  1 54 
tests  for,  152-157 
trichloracetic  acid  test  in,  153 
Tsuchiya's  estimation  of  albu- 
min, 157 
Ulrich's  test,  155 
Alimentary  glycosuria,  161 
Alkaline  phosphates  in  urine,  129 
urine,    unorganized    sediments 
in,  202,  203 
Alkalinity,  fixed,  of  urine,  107 

volatile,  of  urine,  107 
Alkapton  bodies  in  urine,  183 
Alkaptonuria,  183 
All-glass  syringe,  Luer,  for  serum- 
work,  604 
Aluminum  pressure  cooker,  559 
Alveolar  cells  in  sputum,  93 
Amboceptor,  602 

antisheep,  for  Wassermann  re- 
action, 620 
in  Wassermann  reaction,  titra- 
tion of,  624 
Ameboid  motion,  454 
Ammonia  in  urine,  145 
Brown's  test,  147 
decreased,  146 
estimation,  quantitative,  146 
by     Folin    and    Denis' 

method,  147 
by  Ronchese-Malfatti 
method,  147 
increased,  146 
Aramoniacal     decomposition     of 

urine,  106 
Ammoniated  silver  nitrate  solu- 
tion, 143,  144 
Ammonio-magnesium  phosphate 

crystals  in  urine,  211 
Ammonium     sulphate     test     for 
globulin  in  cerebrospinal  fluid, 

525 
urate  crystals  in  urine,  215 
Amorphous  phosphates  in  urine, 
105,  129,  214 
urates  in  urine,  105,  142,  205 
in  mass,  226 
Amylase  in  feces,  434 
estimation,  435 
in  urine,  r48 


Amylase    test  for   pancreatic  in- 
sufficiency, 148,  435 
Anaemia   infantum  pseudoleukae- 

mica,  389 
Anaerobic      bacteria,       cultural 

methods,  579 
Ancylostoma  duodenale,  501 
Anemia,  380 
aplastic,  384 
blood-plaques  in,  301 
pernicious,  383 

color  index  of  blood  in,  284 
degeneration  of  Grawitz  in, 

319 
erythroblasts  in,  323 
leukopenia  in,  287 
lymphocytes  in,  327 
megaloblasts  in,  323 
myelocytes  in,  341 
posthemorrhagic,  382 
primary,  383 
secondary,  381 
splenic,  386 
von  Jaksch's,  389 
Anemias,  degeneration  of  Grawitz 
.in,  319 
Vincent's,  539 
spirochete  of,  462 
Anguillula,  494 
aceti,  494 
in  urine,  237 
AnUin-gentian    violet    stain    for 

bacteria,  640 
Animal  inoculation,  534 

method  for  Bacillus  tubercu- 
losis in  sputum,  81 
of  bacteria,  580 
parasites,  448.     See  also  Para- 
sites, animal. 
Anisocytosis,  316 
Anopheles,  352 
Anthracosis,  sputum  in,  71 
Antibodies,  251 
Antiformin   method   for   Bacillus 

tuberculosis  in  sputum,  80 
Antigen,  600,  602 

in  Wassermann  reaction,  titra- 
tion of,  625 
syphilitic,  in  Wassermann  reac- 
tion, 619 
Antimeningococcus  serum-test  for 
cerebrospinal  meningitis,  530 


INDEX 


657 


Antipyrin  in  urine,  192 
Antisheep  amboceptor  for  Was- 

sermann  reaction,  620 
Anuria,  103 
Aperture,  numeric,  30 
Aplastic  anemia,  384 
Apochromatic  objectives,  26 
Apothecaries'  measure,  table,  653 

weight,  table,  654 
Apparatus,  558,  603,  647 
Arachnoidea,  513 
Arneth's  classification  of  neutro- 

philes,  334_ 
Arsenic  in  urine,  192 

Gutzeit's  test  for,  192 
Reinsch's  test  for,  192 
Arthropoda,  511 
Ascaris,  495 

canis,  496 

lumbricoides,  495 

mystax,  496 
Asexual  cycle  of  malarial  para- 
sites, 349 
Aspirin  in  urine,  198 
Asthma,    bronchial,    eosinophil ia 

in>  337 
sputum  in,  97 
Atrophic    gastritis,   gastric    con- 
tents in,  418 
Atropin  in  urine,  193 
Autoclave,  559 
Autogenous  vaccines,  585 
Avoirdupois  weight,  table,  653 


Babcock    estimation    of    fat    in 

milk,  546 
Babesia,  471 

bigeminum,  47    - 
hominis,  47 
Bacillus,  acid-fast,  82 

Boas-Oppler,    in    gastric    con- 
tents, 416 
colon,  583 
diphtheria,  583 

Neisser's  stain  for,  538 
•       Ponder's  stain  for,  538 
fusiform,  462,  539 
influenza,  584 

in  sputum,  89 
Koch-Weeks,  in  conjunctivitis, 
540 

42 


Bacillus    mucosus  capsulatus    in 
sputum,  88 
of  Friedliinder  in  sputum,  88 
pertussis  in  sputum,  90 
pyocyaneus  in  otitis,  543 
tuberculosis,  584 

fuchsin  stain  for,  639 

in  cerebrospinal  fluid,  531 

in  ear,  543 

in  feces,  442 

in  sputum,  76 

animal  inoculation  method, 

81 
antiformin  method,  80 
Gabbett's  method  for,  77 
LoflSer's  method,  81 
Pappenheim's  method  in, 

Ziehl-Neelson  method  for, 
78 
in  urine,  235 

detection  of,  236 
Pappenheim's   methylene 
blue  for,  641 
typhoid,  583 
in  blood,  346 
technic,  346 
xerosis  in  eye,  542 
Bacteria,  agglutination  of,  607 
anaerobic,     cultural    methods, 

579 
anilin-gentian  violet  stain  for, 

640 
animal  inoculation  of,  580 
carbol-fuchsin  test  for,  639 
carbol-thionin  stain  for,  639 
characteristics  of,  580 
cultural  methods,  577 
cultures  of,  study,  578 
Czaple wski 's  carbol -gentian 

violet  stain  for,  640 
direct  microscopic  examination, 

577 
formalin-gentian    violet    stain 

for,  640 
gentian  violet  stain  for,  640 
Gram-negative,  573 
Gram-positive,  573 
Gram's  stain  for,  572 
Harris'  hematoxylin  stain  for 

640 
hematoxylin  stain  for,  640 


658 


INDEX 


Bacteria  in  blood,  345 
technic  for,  347 

in  cerebrospinal  fluid,  531 

in  conjunctivitis,  540 

in  feces,  441 

in  gastric  contents,  416 

in  milk,  544 

in  otitis  media,  543 

in  sputum,  75 

in  urine,  105,  234 

iodine  stain  for,  641 

Loeffler's     alkaline    methylene 
blue  for,  641 

methods  of  studying,  576 

obtaining,  for  vaccines,  587 

Pappenheim's  pyronin-methyl- 
green  stain  for,  642 
Bacterial  casts  in  urine,  224 

stains,  simple,  642 

vaccines,   585.     See   also    Vac- 
cines. 
Bacterins,    585.     See    also    Vac- 
cines. 
Bacteriologic  methods,  558 
Balantidium,  471 

coli,  471 

minutum,  472 
Basket-cells,  343 

Basophilic  granular  degeneration, 
318 

leukocytes,  338 

stippling,  318 

structures  of  blood,  307 
Bass    and    Johns'     method     for 

malarial  parasites,  359 
Bass  and  Watkins'  modification 

of  macroscopic  Widal  reaction, 

608. 
Bass'  ruling  for  count  in  leukemia, 

296 
Beef  extract  bouillon,  preparation 
of,  565 

infusion,  preparation  of,  564 

tapeworm,  482 
Bence- Jones'  protein  in  urine,  158 
Benedict's  estimation  of  glucose 
in  urine,  167 

test  in  glycosuria,  163 
Benzidin  test  for  blood,  365 

for  hemoglobin,  182 
B.  E.  tuberculin,  595 
B.  F.  tuberculin,  595 


Bial's  orcinol  test  for  pentose  in 

urine,  172 
Bichromate  cleaning  fluid,  563 
Bile  acids  in  urine,  detection,  180 
Hay's  test  for,  180 

diminished  flow  of,  indican  in 
urine  from,  133 

in  feces,  430 

in  gastric  contents,  399 

in  urine,  178 

Smith's  test  for,  179 

medium,  569 
Bile-pigment  in  urine,  detection 

of,  179 
Bilharzia  haematobia,  477 
Bilharziasis,  477 
Biliousness,  indican  in  urine  in, 

.^33  . 

Biologic     identification     of     un- 
known proteins,  611 

Black  sputum,  60 

Bladder,  hemorrhage  from,  234 
Schistosomum    haematobium 
as  cause,  234 

Blood,  249 

acidophilic  structures  of,  307 
agar-agar,  preparation  of,  566 
agglutination,  testing,  375 
animal  parasites  in,  348 
Bacillus  typhosus  in,  346 

technic,  346 
bacteria  in,  345 

technic  for,  347 
basophilic  structures  of,  307 
benzidin  test  for,  365 
carbol-thionin  stain  for,  314 
changes  in,  albuminuria  from, 

chemic  examination,  372 
coagulation,  257 

Bogg's  method  of  estimating, 
258 

prevention,  257 

time,  258 
color,  251 

index  of,  284 
composition  of,  249 
crisis,  383 
diseases,  blood  change  in,  table, 

391 
Ehrlich's  triple  stain  for,  308 
eosinophilic  structures  of,  307 


INDEX 


659 


Blood,  erythrocytes  in,  249 
filarial  larvae  in,  362 
for  transfusion,  matching,  375 
gametes  in,  in  malaria,  351 
Giemsa's  stain  for,  313 
guaiac  test  for,  364 
hematoxylin    and    eosin    stain 

for,  307 
hemin  test  for,  365 
hemoconion  in,  250 
in  acute  myelogenous  leukemia, 
•       388 
in   anaemia   infantum   pseudo- 

leuksemica,  389 
in  anemia,  380 

aplastic,  384 . 

posthemorrhagic,  382 

primary,  383 

progressive  pernicious,  383 

secondary,  381 

splenic,  386 
in  aplastic  anemia,  384 
in  chlorosis,  385 
in  feces,  429 
in  gastric  contents,  399,  407 

test  for,  407 
in  leukemia,  387 

lymphatic,  389 
"myelogenous,  387 
in     posthemorrhagic     anemia, 

382 
in  primary  anemia,  383 
in   progressive   pernicious  ane- 
mia, 383 
in  secondary  anemia,  381 
in  splenic  anemia,  386 
in  sputum,  59 
in   urine,    105,    232,    233.     See 

also  Hematuria. 
Jenner's  stain  for,  313 
larvae  of  Trichinella  spiralis  in, 

363 
leukocytes  in,  250 
malarial  parasites  in,  349.     See 

also  Malarial  parasites. 
neutrophilic  structures  of,  307 
obtaining,  252 

for  Widal  reaction,  606 

from  vein,  254 
oxyphilic  structures  of,  307 
Pappenheim's    panoptic    stain 

for,  313 


Blood  parasites,  345 
pathology,  special,  380 
polychrome  methylene-blue- 

eosin  stains  for,  309,  312 
reaction,  251 

recognition  of,  tests  for,  364 
spectroscopic  test  for,  366 
Teichmann's  test  for,  365 
total  amount,  251 
transfusion,   grouping   individ- 
uals for,  376,  379 
typhoid  bacilli  in,  346 

technic,  346 
unstained,    malarial    parasites 

.in,  355 
viscosity  of,  379 
vital  staining,  373 
volume  index  of,  285 

Lansbee's  method,  286 
watery,  251 
Wright's  stain  for,  309 
application,  310 
preparation,  310 
Blood-casts  in  urine,  223 
Blood-cells,  oxydase  test  for,  342 
Blood-corpuscles,  red.     See  Ery- 
throcytes. 
Blood-corpuscles,   white.      See 

Leukocytes. 
Blood-dust  of  Muller,  250 
Blood-films,  chemic  fixation,  306 
cigarette-paper  method,  304 
Ehrlich's     two   cove  r-g  lass 

method,  303 
fixing,  306 
heat  fixation,  306 
Kowarsky's  plate  for  fixation, 

306 
making,  302 

malarial  parasites  in,  357 
spreading,  302 
stained,  study  of,  302,  314 
staining,  302,  307 
two-slide  method,  304 
Blood-lancet,  Daland's,  252 
Blood-plaques,  250 
enumeration,  300 
in  anemia,  301 
in  infections,  300 
in  leukemia,  301 
in  purpura  hemorrhagica,  301 
in  tuberculosis,  301 


66o 


INDEX 


Blood -plaques,  enumeration, 
Wright  and  Kinnicutt's 
method,  301 

stained,  study  of,  344 
Blood-platelets,  250 
Blood-serum,  251,  257 

antibodies  in,  251 

LofBer's,  preparation  of,  567 
Blood-stain,  Wright's,  for  Trepo- 
nema pallidum,  551 
Boas'  reagent,  401 

test  for  free  hydrochloric  acid, 
401 

test-breakfast,  394 
Boas-Oppler    bacillus    in    gastric 

contents,  416 
Bodies,  alkapton,  in  urine,  183 

Cabot's  ring,  323 

Howell- Jolly,  321 

inclusion,  Dohle's,  335 

Leishman-Donovan,  466 

trachoma,  542 
Bodo,  467 

urinarius,  467 
Boggs'    coagulation    instrument, 

259 
method    of    estimating    blood 

coagulation,  258 
modification   of  Esbach's  esti- 
mation of  milk  proteins,  547 
throttle     control     for     blood- 
counting  pipet,  297 
Boil,   Delhi,  Leishmania  tropica 

of,  466 
Borchardt's  test  for  levulose,  171 
Boric  acid  in  milk,  Goske's  test 

for,  548 
Boston's  method  of  transporting 

semen,  553 
Bottles  for  vaccines,  586 
Bouillon,   beef  extract,  prepara- 
tion of,  565 
infusion,  preparation  of,  565 
Brick-dust  deposit  in  urine,  105 
Bromids  in  urine,  193 
Bronchi,  cylindric  cells  from,  in 

sputum,  93 
Bronchial  asthma,  eosinophilia  in, 
337 
sputum  in,  97 
casts  in  sputum,  61 
spirochetosis,  463 


Bronchiectasis,  sputum  in,  96 

Bronchitis,  acute,  sputum  in,  95 
chronic,  sputum  in,  95 

Brood  membrane,  485 

Brown  sputum,  60 

Brown's  test  for  ammonia  in 
urine,  147 

Bubbles  in  oil-immersion  objec- 
tive, 29 

Buerger's  method  for  pneumo- 
coccus  capsules,  87 

Burker's  hemacytometer,  281 

Butyric  acid  test,  Noguchi's,  for 
globulin  in  cerebrospinal  fluid, 
52s 

Cabot's  ring  bodies,  323 
Calcium  carbonate  in  urine,  214 

oxalate  in  urine,  205    «« 
Calculus,  renal,  urine  in,  244 

vesical,  urine  in,  247 
Calmette's  ophthalmo-tuberculin 

reaction,  596 
Camera  for  microphotography,  46 
Cane-sugar  in  urine,  171 
Capillary  pipets  for  serum- work, 

604 
Capsules,  glass,  for  serum-work, 
604 
pneumococcus,       Buerger' s 
method  for,  87 
Rosenow's  method  for,  88 
Smith's  method  for,  87 
Wright,  562 
Carbol-fuchsin  test  for  bacteria, 

^39  .        . 

Carbol-gentian  violet  stain  (Czap- 

lewski's)  for  bacteria,  640 
Carbol-methyl  violet  stain,  84 
Carbol-thionin  stain  for  bacteria, 

639 
for  blood,  314 
Carbon  monoxid  hemoglobin,  260 

tests  for,  261,  372 
Carbon-laden  cells  in  sputum,  71 
Carcinoma,  gastric,  gastric  con- 
tents in,  418 
Cardboard  micrometer,  44 
Carriers,  dysentery,  454 
Casts,  bronchial,  in  sputum,  61 
fatty,  in  urine,  222 
fibrinous,  in  urine,  221 


INDEX 


66i 


Casts,  granular,  in  urine,  222 
hyaline,  in  urine,  219 
tube-,  in  urine,  216 

examination  for,  218 
waxy,  in  urine,  221 

Catarrh,      vernal,      eosinophilic 
leukocytes  in,  542 

Cells,  alveolar,  in  sputum,  q3 
basket,  343 

carbon-laden,  in  sputum,  71 
cylindric,  in  sputum,  93 
eosinophilic,  in  sputum,  gr 
epithelial,  in  feces,  440 
in  sputum,  92 
in  urine,  227 
heart-failure,  in  sputum,  70 
in  sputum,  90 
irregular,  in  urine,  228 
mast-,  338 

mesothelial,  predominance,  523 
pavement,  in  urine,  229 
pigmented,  in  sputum,  70 
polyhedral,  in  urine,  227 
pus-,  in  gastric  contents,  415 
shadow,  in  urine,  233 
squamous,  in  sputum,  92 

in  urine,  229 
vegetable,  in  feces,  428,  438 
yeast-,  in  gastric  contents,  415 
in  urine,  239 

Cement,  shellac,  39 

Centigrade  and  Fahrenheit  scales 

654 
Central   illumination   for   micro- 
scope, 22 
Centrifugal  method,  Purdy's,  for 
albumin  in  urine,  158 
of  examination  of  urine,  120 
Centrifuge  for  serum-work,  603 
Purdy  electric,  for  urine,  121 

122 
tubes,  Purdy's,  122 
water-motor,  for  urine,  121 
Cercomonas,  467 

hominis,  467 
Cerebrospinal  fluid.  Bacillus  tu- 
berculosis in,  531 
bacteria  in,  531 
cytology  of,  532 
examination,  524 
chemic,  525 
macroscopic,  524 


Cerebrospinal  fluid,  examination, 
microscopic,  531 
globulin  in,  525 

ammonium  sulphate  test, 

52s 
Noguchi's  butyric  acid  test, 

525 
Pandy's  test,  526 
Lange's    colloidal    gold    test 
for,  526 
preparation    of    re- 
agent, 527 
technic,  528 
lymphocytes  in,  533 
mastic  test  for,  528 

preparation  of  solutions, 

529 
technic,  530 
sugar  in,  530 

Wassermann   reaction   with, 
628 
meningitis,  epidemic,  531 

antimeningococcus-serum 
test  for,  530 
Cestoda,  473,  480 
Cestodes,  480 

Charcot-Leyden  crystals  in  feces, 
442 
in  urine,  69 
Chemic  fixation  of  blood-films,  306 
Chemotaxis,  288 
Chloral  hydrate  in  urine,  193 
Chlorids  in  urine,  124 
detection,  126 

estimation,      centrifugal 
method,  129 
quantitative,  126 
table  for,  128 
Volhard's  method,  127 
Chlorosis,  385 

lymphocytes  in,  327 
Chrysomyia  macellaria,  513 
Chyluria,  211 

Cigarette-paper  method  for  blood- 
films,  304 
Ciliata,  453,  471 
Clarification  of  urine,  loi 
Cleaning  fluid,  bichromate,  563 
Coagulation  of  blood,  257 
Cobra-venom  test  for  syphilis,  636 
materials  required,  636 
technic,  637 


662 


INDEX 


Coccidium,  470 
cuniculi,  470 
Cochin  China  diarrhea,  506 
Cofiin-lid  crystals  in  urine,  211 
Coin-like  disks  in  sputum,  98 
Colloidal   gold    test,   Lange's    of 
cerebrospinal  fluid, 

526 
preparation    of    re- 
agent, 527 
technic,  528 
Colon  bacillus,  583 
Color  index  of  blood,  284 

in  pernicious  anemia,  284 
of  urine,  103 
Colorimeter,  Denison  Laboratory, 
119 
Hellige,  117 
Kuttner,  118 
Colorimetric  methods  of  examina- 
tion of  urine,  116 
Complement,  602 

deviation    test   for  gonorrhoea, 
632 
for  syphilis,  619 
for  tuberculosis,  633 
in  Wassermann  reaction,  621 
titration  of,  623 
Concentration  methods  for  mala- 
rial parasites,  359 
Concretions  in  feces,  427 
Condenser  for  microscope,  20,  25 
Congo-red  test  for  free  acids  in 

gastric  contents,  400 
Conjugate  sulphates  in  urine,  132 
Conjunctivitis,   acute   infections, 
540 
bacteria  in,  540 
diphtheric,  542 
pseudomembranous,  542 
Contact    tests    for    albuminuria, 

152 
Cooker,  aluminum  pressure,  559 
Cook's  method  of  estimating  uric 

acid  in  urine,  143 
Corpuscles,  pus-,  33i>  SiS 
red  blood-.     See  Erythrocytes. 
white.     See  Leukocytes. 
Cotton  fibrils  in  sputum,  66 

sterilization  of,  563 
Counting  of  vaccines,  589 
Craig's  test  for  tuberculosis,  634 


Crenation  of  red  corpuscles,  2i;o 

Crisis,  blood,  383 

Croupous  pneumonia,  sputum  in, 

60,  97 
Crystals,  ammonio-m  agnesium      / 
phosphate,  in  urine,  211    — .^/ 
ammonium  urate,  in  urine,  215 
Charcot-Leyden,  in  feces,  442 

in  urine,  69 
cystin,  in  urine,  288 
dicilcium  phosphate,  in  urine, 

213 
envelope,  in  urine,  205 
in  feces,  442 
in  sputum,  69 
leucin,  in  urine,  208 
small,  in  urine,  226 
thorn-apple,  in  urine,  215 
tyrosin,  in  urine,  208 
uric-acid,  in  urine,  203 
Culex,  353 
Cultural  methods,  577 

for  anaerobic  bacteria,  579 
media,  564 
inoculating,  method  of,  577 
preparation,  564 
reaction  of,  570 
sterilization  of,  562 
storage  of,  571 
tubing  of,  571 
Cultures  of  bacteria,  study  of,  578 
Culture-tubes,  560 
plugging  of,  564 
preparation  of,  563 
Curds  in  feces,  428 
Curschmann's  spirals  in  sputum, 

Curvature  of  microscopic  field,  29 
Cutaneous  test  for  syphilis,  598 
Cyanosis,  enterogenous,  260 
Cyclic  albuminuria,  150 
Cylindric  cells  in  sputum,  93 
Cylindroids  in  urine,  226 
Cylindruria,  216 
Cysticercus  cellulosae,  485 
Cystin  crystals  in  urine,  208 
Cystinuria,  209 
Cystitis,  urine  in,  246 
Cysts,  daughter-,  485 
Cytodiagnosis,  521 
Cytology   of  cerebrospinal   fluid, 
532 


INDEX 


663 


Czaplewski's  carbol-fuchsia  stain 

for  bacteria,  639 
Czaplewski's  carbol-gentian  violet 

stain  for  bacteria,  640 

Daland's  blood-lancet,  252 

hematocrit,  286 
Dare's  hemoglobinometer,  268 
Dark-ground  illumination  for  mi- 
croscope, 24 
for  Treponema  pallidum,  552 
Daughter-cysts,  485 
Decomposition  of  urine,  100 

ammoniacal,  106 
Degeneration,   basophilic   granu- 
lar, 318 
of  Grawitz,  318 
Delhi  boil,  Leishmania  tropica  of, 

466 
Demodex  folliculorum,  5 1 1 
Denison  Laboratory  colorimeter, 

119 
Deposit,  brick-dust,  in  urine,  105 
Desmoid  test,  Sahli's,  of  gastric 

digestion,  421 
Dextrose  in  urine,  161.     See  also 

Glycosuria. 
Diabetes  insipidus,  urine  in,  248 

mellitus,  urine  in,  248 
Diacetic  acid  in  urine,  177 
detection,  177 
Gerhardt's  test,  177 
Diagnosticum,  typhoid,  of  Ficker, 

614 
Diarrhea,  Cochin  China,  506 
Diazo  reaction  in  measles,  189 
in  tuberculosis,  189 
in  typhoid  fever,  188 
substitutes  for,  190 
technic,  189 
substances  in  urine,  188 
Dibothriocephalus,  490 
latus,  481,  490 
anemia  from,  382 
Dicalcium  phosphate  crystals  in 

urine,  213 
Dicroccelium,  475 
lanceatum,  475 
Digestion,  stomach,  392 
Digestive  leukocytosis,  290 
Dilatation    of    stomach,    gastric 
contents  in,  417 


Diluting   fluids   for  blood-count, 
279 
in  leukemia,  300 
vaccines,  591 
Dimethylamidoazobenzol  test   or 

free  hydrochloric  acid,  400 
Diphtheria  bacillus,  583 
Neisser's  stain  for,  538 
Bonder's  stain  for,  538 
Schick  test  in,  599 
Diphtheric  conjunctivitis,  542 
Diplococcus,    Frankel's,    in    spu- 
tum, 85 
intracellularis  meningitidis,  531, 
583 
Diplococcus  of   Morax-Axenfeld, 

54°. 
Dipylidium,  489 

caninum,  489 
Dirt  on  cover  glass  as  source  of 

error,  241 
Disks,  coin-like,  in  sputum,  98 
Dittrich's  plugs  in  mucus,  61 
Dohle's  inclusion  bodies,  335 
Doremus- Hinds  ureo meter,  137 
Dourine,  trypanosome  of,  465 
Drugs  in  urine,  191 

leukocytosis  from,  292 

resinous,  in  urine,  198 
Drunkard's   pneumonia,   sputum 

in,  60 
Dry  objective,  28 

sterilizer,  558 
Duke's    coagulation  instrument, 

258- 
Dunham's  peptone  solution,  prep- 
aration of,  569 
Dwarf  tapeworm,  487 

Ear,  bacteria  in,  543 

diseases  of,  543 
Earthy  phosphates  in  urine,  129 
Echinococcus  disease,  485 

diagnosis,  486 

eosinophilia  in,  337 
Edema,  pulmonary,  sputum  in,  96 
Edestin  test  for  gastric  contents, 

407 
Eel,  vinegar,  494 

in  urine,  237 
Egg  medium,  preparation  of,  568 
Egyptian  hematuria,  234,  477 


664 


INDEX 


Ehrlich's  diazo  reaction,  i88 
substitutes  for,  190 
technic,  189 
side-chain  theory  of  immunity, 

600 
test  for  urobilinogen,  186 
triple  stain  for  blood,  308 
two-cover-glass  method  foi 
blood-films,  303 
Einhorn's  saccharimeter,  168 
Elastic  fibers  in  feces,  440 

in  sputum,  64 
Electric  lamp  for  microscope,  19 
Empty  magnification,  34 
EndamcEba,  453 
buccalis,  457 
coli,  458 

E.  histolytica  and  differentia- 
tion, 456,  457 
dentalis,  457 
gingivalis,  457 

in  pyorrhea  alveolaris,  459 
histolytica,  453 

ameboid  motion,  454 

E.    coli   and   differentiation, 

456,  457 
in  sputum,  74 
tetragena,  459 
Endocarditis,  malignant,  vaccines 

in,  593 
Endogenous  uric  acid  in  urine,  142 
Endomyces  albicans,  537 
Endothelial  leukocytes,  329 
Endotheliocytes,  329 
Endotin,  595 

Enterogenous  cyanosis,  260 
Enteroliths  in  feces,  427 
Envelope  crystals  in  urine,  205 
Enzyme,  peptid-splitting,  in  gas- 
tric contents,  405 
Eosinophiles,  336 

predominance,  523 
Eosinophilia,  337 
in  bronchial  asthma,  337 
in  echinococcus  disease,  337 
in  filariasis,  337 
in  menstruation,  337 
in  myelogenous  leukemia,  337 
in  scarlet  fever,  337 
in  skin  diseases,  337 
in  trichiniasis,  337 
in  uncinariasis,  337 


Eosinophilic  cells  in  sputum,  91 

leukocytes,  336 

in  vernal  catarrh,  542 

structures  of  blood,  307 
Epidemic  cerebrospinal  meningi- 
tis, 531 
antimeningococcus-serum 
test  for,  530 
Epithelial  casts  in  urine,  223,  227 

cells  in  feces,  440 
in  sputum,  92 
Equilibrium,  nitrogen,  135 
Equipment  for  office  laboratory, 

643 
Erythroblasts,  320 

in  pernicious  anemia,  323 
Erythrocytes,  249 

agglutination  of,  376 

basophilic    granular    degenera- 
tion of,  318 

Barker's  instrument  for  count- 
ing, 281 

Cabot's  ring  bodies  in,  323 

counting  of,  272 

crenation  of,  250 

decrease  of,  271 

diluting  fluid  for  counting,  279 

enumeration,  270 

fragility  of,  374 

hemoglobin  in,  316 

in  feces,  441 

in  gastric  contents,  414 

in  leukemia,  317 

in  pernicious  anemia,  317 

in  sputum,  94 

in  urine,  232 

increase  of,  270 

malarial  stippling  in,  319 

nuclear  particles  in,  321 

nucleated,  significance  of,  322 

pessary  forms,  316 

resistance,  test  for,  374 

rouleaux  formation  of,  249 

shape  of,  316 

sheep's,   in  Wassermann  reac- 
tion, 620 

size  of,  316 

stained,  study  of,  315 

staining  power,  variations  in, 

317 
Thoma-Metz    instrument    for 
counting,  283 


INDEX 


66: 


Erythrocj'tes,     Thoma-Zeiss    in- 
strument for  counting,  273 
variations  in  structure,  320 
Esbach's  albuminometer,  157 
estimation    of    milk    proteins, 

Bogg's  modification,  547 
method  for  estimating  albumin 

in  urine,  157 
reagent  for  albuminuria,  157 
Estivo-autumnal    malarial    para- 
sites, 34Q,  350,  356 
Ethereal  sulphates  in  urine,  132 
Ewald's    salol    test    for    gastric 
motility,  420 
test-breakfast,  394 
Exhausting  diseases,  anemia  from, 

381 
Exogenous  uric  acid  in  urine,  142 
Extraneous   structures   in   urine, 

239 
ExOdates,  520 
Eye,  540 

pink-,  540 
Eye-piece  for  microscope,  26 

micrometer,  for  microscope,  42 
step,  43 

Fahrenheit      and      Centigrade 

scales,  654 
False  albuminuria,  150 
Fasciola,  474 

hepatica,  474 
Fasciolopsis,  476 

buski,  476 
Fasting  gastric  contents,  398 
Fat  in  feces,  439 

in  milk,  546 

Scharlach  R  stain  for,  643 

Sudan  III  stain  for,  643 
■Fat-droplets  in  urine,  241 
Fat-globules  in  urine,  210 
Fatty  casts  in  urine,  222 
Favus,  543 
Feces,  423 

acholic,  425 

amylase  in,  434 
estimation,  435 

animal  parasites  in,  427 

Bacillus  tuberculosis  in,  442 

bacteria  in,  441 

bile  in,  430 

blood  in,  429 


Feces,    Charcot-Leyden    crystals 
in,  442 

chemic  examination,  429 

color,  424 

concretions  in,  427 

consistence,  424 

crystals  in,  442 

curds  in,  428 

elastic  fibers  in,  440 

enteroliths  in,  427 

epithelial  cells  in,  440 

erythrocytes  in,  441 

examination,  chemic,  429 
macroscopic,  424 
microscopic,  436 

food  particles  in,  437 

form,  424 

frequency,  424 

functional  tests,  444 
motility,  447 
Sahli's  glutoid,  446 
Schmidt's  diet,  444 
nuclei,  446 

gall-stones  in,  427 

hydrobilirubin  in,  184,  430 

macroscopic  examination,  424 

microscopic  examination,  436 

mucus  in,  426 

muscle-fibers  in,  438 

normal  contents,  423 

occult    hemorrhage    in,    detec- 
tion, 429 

odor  of,  425 

ova  in,  443 

pancreatic  ferments  in,  434 

parasites  in,  443 

pus  in,  440 

quantity,  424 

soaps  in,  440 

starch-granules  in,  438 

trypsin  in,  434 
test  for,  436 

urobilin  in,  430.     See  also  Uro- 
bilin in  feces. 

vegetable  cells  in,  428,  438 
fibers  in,  428,  438 
hairs  in,  438 
Fehling's  estimation  of  glucose  in 
urine,  166 

test  for  glycosuria,  163 
Ferment  diagnosticum,  406 
Fermentation,  acid,  of  urine,  106 


666 


INDEX 


Fermentation  method  for  glucose 
in  urine,  169 
test  for  glucose,  165 
Ferments,    pancreatic,    in    feces, 

.434 
Fibers,  elastic,  in  feces,  440 
in  sputum,  64 

in  urine,  227 

muscle,  in  feces,  438 
in  urine,  242 

textile,  in  urine,  241 

vegetable,  in  -feces,  428,  438 
Fibrils,  cotton  in  sputum,  66 
Fibrinous  casts  in  urine,  221 
Ficker's    typhoid    diagnosticum, 

614 
Filaria,  498 

bancrofti,  498 

loa,  500 

medinensis,  500    . 

perstans,  500 

philippinensis,  500 

sanguinis  hominis,  499 
Filarial  larvae  in  blood,  362 
Filariasfs,  498 

diagnosis,  499 

eosinophilia  in,  337 
Filariform  larvae,  508 
Films,  blood.     See  Blood-films. 
Fischer's  test-meal,  395 
Fish  tapeworm,  490 
Fixation  of  blood-films,  306 
by  heat,  306 
chemic,  306 
Flagella,  LofHer's  stain  for,  575 
Flasks,  560 
Flat  worms,  473 
Flaws  in  slide  as  source  of  error, 

241 
Fleischl-Miescher  hemoglobinom- 

eter,  266 
Floaters,  gonorrheal,  in  urine,  519 

in  urine,  237 
Florence's  reaction  for  semen,  554 
Flukes,  474 

liver,  474 

lung,  476 

worm,  473 
Focus,  depth  of,  34 
Folin  and  Denis'  method  of  esti- 
mating ammonia  in  urine, 
147 


Folin  and  Denis'  urease  method 

for  examining  urine,  140 
Folin's    method    of    quantitative 
estimation  of  acidity  of  urine, 
107 
Food  particles  in  feces,  437 

in  gastric  contents,  399,  414 
Formaldehyd  in  milk,  548 
in  urine,  Rimini-Burnam  test 
for,  194 
Formalin  in  milk,  test  for,  547 
method  of  estimating  ammonia 
in  urine,  147 
Formalin-gentian  violet  stain  for 

bacteria,  640 
Fractional  method  for  gastric  con- 
tents, 397 
Frankel's  diplococcus  in  sputum, 

■85 
Frommer's  test  for  acetone,  177 
Frothingham's  method  for  Negri 

bodies,  555 
Fruit  sugar  in  urine,  170 
Fuchsin  stain  for  bacteria,  639 
Functional  tests  for  feces,  444 
motility,  447 
Sahli's  glutoid,  446 
Schmidt's  diet,  444 
nuclei,  446 
for  urine,  112 
intestines,  motility,  447 
kidney,  phenolsulphonphtha- 

lein  test,  112 
liver,Strausslevulose  test,i7o 

urobilin,  185 
pancreas,  amylase,  148,  435 
Sahli's  glutoid  test,  446 
Schmidt's  nuclei  test,  446 
trypsin,  436 
stomach,   absorptive   power^ 
419 
Sahli's  desmoid  test,  421 
motor  power,  420 
Fungi,  mold,  in  urine,  240 
Fungus,  thrush,  537 
Furniture  for  laboratory,  645 
Fusiform  bacillus,  462,  539 

Gabbett's    method   for   Bacillus 
tuberculosis  in  sputum,  77 
stain  for  Bacillus  tuberculosis, 
641 


INDEX 


667 


Gafifky's  table  for  recording  num- 
ber of  tubercle  bacilli  in  sputum, 
80 
Gall-stones  in  feces,  427 
Gametes  in  blood  in  malaria,  351 

detection,  358 
Gangrene  of  lung,  sputum  in,  96 
Gastric   carcinoma,   gastric   con- 
tents in,  418 
contents,  bacteria  in,  416 
bile  in,  399 
bits  of  tissue  in,  39.9 
blood  in,  399,  407 

test  for,  407 
Boas-Oppler  bacillus  in,  416 
chemic  examination,  400 
edestin  test  for,  407 
erj^throcytes  in,  414 
examination,  chemic,  400 

fractional,  397 

microscopic,  414 

phj'sical,  398 

routine,  393 
fasting,  398 

food  particles  in,  399,  414  ' 
fractional  examination,  397 
free  acids  in,  tests  for,  400 

Congo-red  test,  400 
hydrochloric    acid    in,    393. 

See  also  Hydrochloric  acid, 

free. 
in  achylia  gastrica,  418 
in  atrophic  gastritis,  418 
in  chronic  gastritis,  418 
in  dilatation,  417 
in  disease,  417 
in  gastric  carcinoma,  418 
in  neuroses,  417 
in  ulcer,  419 
lactic  acid  in,  401.     See  also 

Lactic  acid. 
Leptothrix  buccalis  in,  416 
mucus  in,  399 
obtaining,  393 
organic  acids  in,  401,  412 
pepsin  in,  403.     See  also  Pep- 
sins in  gastric  contents. 
pepsinogen  in,  403 

test  for,  404 
peptid-splitting    enzyme    in, 

40s 
physical  examination,  398 


Gastric  contents,  pus-cells  in,  415 
reaction,  398 
rennin  in,  405 
test  for,  405 
renninogen  in,  405 
routine  examination,  393 
sarcinas  in,  415 
tests,  qualitative,  400 

quantitative,  408 
total  acidity,  408 
tests,  408 
Topfer's  test,  408 
withdrawal,  322 
yeast-cells  in,  415 
digestion,  Sahli's  desmoid  test, 

421 
neuroses,  stomach  contents  in, 

417 
ulcer,  gastric  contents  in,  419 
Gastritis,   atrophic,   gastric   con- 
tents in,  418 
chronic,  gastric  contents  in,  418 
Gauze,  sterilization  of,  563 
Gelatin  media,  preparation  of,  567 
Generator,  hydrogen  sulphid,  195 
Gentian-violet  stain  for  bacteria, 

640 
Gerhardt's  test  for  diacetic  acid, 

.177 
Giemsa's  stain  for  blood,  313 

for  Treponema  pallidum,  550 
Glass  capsules   for   serum-work, 

604 
Glassware,  sterilization  of,  562 
Globular  sputum,  98 
Globules,  fat-,  in  urine,  210 

myelin,  in  sputum,  71 
Globulin   in    cerebrospinal    fluid, 

525 
ammonium   sulphate    test 

for,  525 
Noguchi's     butyric     acid 

test,  525 
Pandy's  test,  526 
Glossina  morsitans,  465 

palpalis,  349 
Glucose  in  urine,  161.     See  also 
*  Glycosuria. 

Glutoid  test,  Sahli's,  for  examina- 
tion of  feces,  446 
Glycerin    agar-agar,    preparation 
of,  566 


668 


INDEX 


Glycosuria,  alimentary,  i6i     ' 
Benedict's  test  in,  163 
detection  of  dextrose,  162 
Fehling's  test  in,  163 
fermentation  test  in,  165 
Haines'  test  in,  162 
Kowarsky's  test  in,  164 
persistent,  161 
phenylhydrazin  test  in,  164 
quantitative  estimation,  165 
Benedict's  method,  167 
Fehling's  method,  166 
fermentation  method,  169 
Roberts'  method,  170 
transitory,  161 
Gmelin's   test   for  bile  in  urine, 

180 
Gold  test,  colloidal,  Lange's,  of 
cerebrospinal 
fluid,  526 
preparation    of    re- 
agent, 527 
technic,  528 
Gonococci  in  pus,  519 

in  urine,  237 
Gonococcus,  582 

in  ophthalmia,  542 
Gonorrhea,  complement  deviation 

test  for,  632 
Gonorrheal  ophthalmia,   542 

threads  in  urine,  225,  237,  519 
Goske's  test  for  boric  acid  in  milk, 

Gram-negative  bacteria,  573 
Gram-positive  bacteria,  573 
Gram's  iodin  solution,  641 

stain  for  bacteria,  572 
Granular  casts  in  urine,  222 

degeneration,  basophilic,    318 
Granules,  Much,  in  sputum,  82 
staining  methods  for,  83 

Schiiffner's,  319 

starch-,  in  feces,  438 
Gravel  in  urine,  203 
Grawitz,  degeneration  of,  318 
Gray  sputum,  60 
Gross'  test  for  trypsin  in  feces, 

436 
Ground  itch,  503 
Guaiac  test  for  blood,  364 

for     hemoglobin     in     urine. 


Guinea-worm,  500 
Gunning's  test  for  acetone,  175 
Gutzeit's  test  for  arsenic  in  urine, 
192 

Haines'  solution,  163 

test  for  glucose  in  urine,  162 
Hairs  in  urine,  227 

vegetable,  in  feces,  438 
Hall's  method  of  estimating  uric 

acid  in  urine,  143 
Hammer's  test  for   tuberculosis, 

633 
Hammerschlag's  test  for  pepsin, 

412 
Harris'    hematoxylin     stain    for 

bacteria,  640 
Hart's  test  for  oxybutyric  acid, 

178 
Haser  method  of  estimating  solids 

in  urine,  in 
Hayem's    fluid    for   blood-count, 
279 

hematoblasts  of,  345 
Hdy's  test  for  bile  acids  in  urine, 

180 
Heart-failure    cells    in    sputum. 

Heat  and  nitric  acid  test  for  al- 
bumin, 156 
fixation  of  blood-films,  306 
test,  Purdy's,  for  albumin,  156 
Hellige  colorimeter,  117 
Hemacytometer,  B  ii  r  k  e  r '  s ,  281 
Levy    counting    chamber    for, 

282 
method   of   counting   vaccines, 

590 
Thoma-Metz,  283 
Thoma-Zeiss,  273 

cleaning  instrument,  280 
sources  of  error,  279 
technic,  273 
Hematemesis,    hemoptysis    and, 

differentiation,  399 
Hematin,  spectroscopic  test  for, 

371 
Hematoblasts  of  Hayem,  345 
Hematocrit,  Daland,  286 
Hematoporphyrin  in  urine, 
184 

spectroscopic  test  for,  371 


INDEX 


669 


Hematoxylin  and  eosin  for  blood, 

307 

stain  for  bacteria,  640 
Harris'  method,  640 
Hematuria,  233 

Egyptian,  234,  477 

hemoglobinuria  and,   differen- 
tiation, 181 

idiopathic,  234 
Hemin  test  for  blood,  365 
Hemochromogen,      spectroscopic 

test  for,  371 
Hemoconion,  250 
Hemoglobin,  260 

carbon  monoxid  in,  260 
test  for,  261 

spectroscopic,  372 

Dare's  estimation,  268 

decrease,  263 

derivatives,  absorption  spectra 
of,  370 

estimation,  264 

Fleischl-Miescher     estimation, 
266 

in  erythrocytes,  316 

in  urine,  180.     See  also  Hemo- 
globinuria. 

increase,  263 

reduced,  spectroscopic  test  for, 
370 

Sahli's  estimation,  266 

spectroscopic  test  for,  370 

Tallqvist's  estimation,  269 

von  Fleischl  estimation,  265 
Hcmoglobinemia,  261 
Hemoglobinometer,  Dare's,  268 

Fleischl-Miescher,  266 

Sahli's,  266,  267 

von  Fleischl's,  264 
Hemoglobinuria,  180 

benzidin  test  for,  182 

detection,  181 

guaiac  test  for,  181 

hematuria  and  differentiation, 
181 

paroxysmal,  181 

spectroscopic  test  for,  183 
Hemolysis,  374 

initial,  375 
Hemolytic  system,  619 
Hemoptysis,    hematemesis    and, 

differentiation,  399 


Hemorrhage,  anemia  from,  381 
from  bladder,  234 

Schistosomum   haematobium 
as  cause,  234 
occult,  in  feces,  detection,  429 
Herpetomonas,  466 
Herxheimer's  Sudan  HI  stain,  643 
Hexamethylenamin  in  urine,  193 
Hip-roof  crystals  in  urine,  211 
Hiss'  serum-water  media,  569 
Holt's  milk-testing  apparatus,  545 
Hookworm,  501 
disease,  503 
diagnosis,  503 
Horismascope,  155 
Hot-air  sterilizer,  558 
Ho wel- Jolly  bodies,  321 
Huntoon's  stain  for  spores,  575 
Hyaline  casts  in  urine,  219 
Hydatid  disease,  485 
Hydrobilirubin  in  feces,  184,  430 
Hydrochloric  acid,  combined,  392, 
410 
deficit,  estimation  of,  411 
free,  334,  393 
absence,  410 
Boas'  test  for,  401 
decrease,  409 
dimethylamidoazobenzol 

test,  400 
increase,  409 

Topfer's  test  for,  410,  411 
Hydrogen  sulphid  generator,  195 
Hydrophobia,555.  See alsoi?aWc5. 
Hymenolepis,  487 
diminuta,  489 
nana,  487 
Hyperchlorhydria,  409 
Hyperchromemia,  263 
Hyperemia,  renal,  urine  in,  242 
Hyphffi  of  molds  in  urine,  227 
Hypobromite  method  of  estimat- 
ing urea  in  urine,  137 
Hypochlorhydria,  409 

Idiopathic  hematuria,  234 
Illumination,     dark-ground,     for 
Treponema  pallidum  552 
for  microscope,  18 
amount  of,  22 

with  water-bottle  condenser, 
20 


670 


INDEX 


Image,  microscopic,  virtual,  30 
Immersion  objective,  25 
Immune  bodies,  601 

of    second    order,  •  reactions 

based  on,  604 
of     third     order,     reactions 
based  on,  6i8 
Immunity,  600 
acquired,  600 
amboceptor,  602 
complement,  602 
Ehrlich's  side-chain  theory,  600 
in  diphtheria,  Schick  test  for, 

599 
receptors,  601,  602 
Incidental  parasites,  472 
Inclusion  bodies  of  Dohle,  335 
Incubator,  559 

for  serum-work,  603 
Index,  color,  of  blood,  284 

opsonic,  615.     See  also  Opsonic 

index. 
phagocytic,  617 
volume,  of  blood,  285, 

Larrabee's  method,  286 
India-ink    stain    for    Treponema 

pallidum,  552 
Indican  in  urine,  132 
detection  of,  133 
from  decomposition  of  exu- 
dates, 133 
from  diminished  flow  of  bile, 

in  biliousness,  133 

in  diseases  of  small  intestine, 

133 
of.  stomach,  133 
Obermayer's  test  for,  134 
Indophenol  oxydase  test  for  mye- 
loblasts, 342 
Infantile     splenomegaly,     Leish- 

mania  infantum  of,  466 
Infections,  enumeration  of  blood- 
pla;ques  in,  300 
leukocytosis  from,  291 
vaccines  in,  593 
Infectious  diseases,  anemia  from, 

3«i 

jaundice,  spirochaete  of,  463 
Inflammations,     acute     pseudo- 
membranous, 537 

leukocytosis  from,  292 


Influenza  bacillus,  584 

Infusion,  beef,  preparation  of,  564 

bouillon,  preparation  of,  565 
Infusoria,  453,  471 
Inoculating     culture-media, 

method  of,  577 
Inoculation,  animal,  534 
of  bacteria,  580 

method,    animal,   for    Bacillus 
tuberculosis  in  sputum,  81 
Inorganic  constituents  of  urine, 

99,  124 
Intestine,  small,  diseases  of,  indi- 

canuria  in,  133 
lodin  solution.  Gram's,  641 

stain  for  bacteria,  641 
lodophilia,  332 
Irregular  cells  in  urine,  228 

malaria,  351 
Irritation     leukocytes,     Tiirck's, 

343 
Iso-agglutinin  groups,  376 
Itch,  ground,  503 

Jaundice,  infections,  spirochaete 

of,  463 
Jenner's  stain  for  blood,  313 

Kala  azar,  Leishmania  donovani 
of,  466 

Keidel's  vacuum  tube  for  collect- 
ing blood,  256 

Kelling's  test  for  lactic  acid,  403 

Kidney,  changes  in,  albuminuria 
from,  151 

Koch-Weeks  bacillus,  in  conjunc- 
tivitis, 540 

Kowarsky's  plate  for  fixing  blood- 
films,  306 
test  for  glucose,  164 

Kuttner  colorimeter,  118 

Laboratory,  apparatus  for,  647 
equipment,  643 
furniture,  645 
Lactic  acid  in  gastric  contents, 
401 
Kelling's  test  for,  403 
Simon's  test  for,  403 
Strauss'  test  for,  403 
Uffelmann's  test  for,  402 


INDEX 


671 


Lactose  in  milk,  estimation,  547 

in  urine,  171 
Lamblia,  469 

intestinalis,  469 
Lamp,  electric,  for  microscope,  19 
Lancet,  Daland's  blood,  252 
Lange's  colloidal  gold  test  of  cere- 
brospinal     fluid, 
526 
preparation  of  rea- 
gent, 527 
technic,  528 
modification  of  Legal's  test  for 
acetone,  176 
Larrabee's  estimation  of  volume 

index  of  blood,  286 
Larvae,  filarial,  in  blood,  362 
filariform,  508 
in  sputum,  74 
of  Trichinella  spiralis  in  blood, 

363. 
rhabditiform,  507 
Lead  in  urine,  194 

Lederer's  test  for,  194 
Lederer's  test  for  lead  in  urine, 

194 
Leflman-Beam  estimation  for  fat 

in  milk,  546 
Legal's  test  for  acetone,  Lange's 

modification,  176 
Leishman-Donovan  bodies,  466 
Leishmania,  466 
donovani,  466 
infantum,  466 
Leishman's   method   for   opsonic 

index,  616 
Leptothrix  buccalis,  535 
in  gastric  contents,  416 
in  sputum,  66 
Leucin  in  urine,  207 
Leukemia,  294,  387 
erythrocytes  in,  317 
in  pernicious  anemia,  317 
leukocyte  count  in,  .294 
lymphatic,  389 

blood-plaques  in,  301 
myelogenous,  387 
acute,  388 
eosinophilia  in,  337 
myelocytes  in,  341 
Leukocytes,  250 

abnormal  varieties,  339 


Leukocytes,  absolute  increase  in, 

325 
atypic  forms,  344 
basophilic,  338 
counting,  differential,  324 
in  leukemia,  294 
size  of  field  required,  299 
decrease  in  number,  287 
degenerated  forms,  343 
endothelial,  329 
enumeration,  287 
eosinophilic,  336 

in  vernal  catarrh,  542 
increase  in  number,  287 
large  mononuclear,  329 
normal  varieties,  326 
polymorphonuclear    neutrophi- 
lic, 330 
predominance,  521 
polynuclear,  330 
relative  increase  in,  325 
transitional,  329 
Tiirck's  irritation,  343 
vacuolated,  343 
Leukocytosis,  287 
digestive,  290 
from  drugs,  292 
from  infections,  291 
from  inflammations,  291 
in  malignant  disease,  292 
lymphocytic,  292 

in  hereditary  syphilis,  293 
in  pertussis,  293,  327 
permanent,  288 
polymorphonuclear,  289 
pathologic,  290 
physiologic,  290 
posthemorrhagic,  292 
toxic,  292 
transient,  288 
Leukopenia,  287 

in  pernicious  anemia,  287 
lymphocytes  in,  327 
Levulose    in    urine,    Borchardt's 
test  for,  171 
detection  of,  170 
quantitative  estimation,  171 
Levy  counting  chamber  for  hema- 
cytometer, 282 
Linguatula  serrata,  513 
Liquor  iodi  compositus,  641 
Litmus  milk,  preparation  of,  568 


672 


nSTDEX 


Liver  fluke,  474 

rot,  475 
LofSer's  alkaline  methj'lcne  blue 
for  bacteria,  641 

blood-serum,    preparation    of, 

567 

method  for  Bacillus  tuberculo- 
sis in  sputum,  81 

stain  for  flagella,  575 
Luer  all-glass  syringe  for  serum 

work,  604 
Luetin,  598 

skin  test  for  syphilis,  598 
Lugol's  solution,  641 
Lung  fluke,  476 

gangrene  of,  sputum  in,  g6 
Lycopodium  as  micrometer,  44 

granules  in  urine,  241 
Lymphatic  leukemia,  389 
Lymphocytes,  326 

forms,  327 

in  cerebrospinal  fluid,  533 

in  chlorosis,  327 

in  leukopenia,  327 

in  pernicious  anemia,  327 

origin,  327 

predominance,  523 
Lymphocytic  leukocytosis,  292 
in  hereditary  syphilis,  293 
in  pertussis,  293,  327 
Lymphocytosis,  293 
Lysins,  602 

Macrocytes,  316 
Magnification  by  microscope,  34 
methods  of  increasing,  36 
empty,  36 
Malaria,  gametes  in  blood  in,  351 
detection,  358 
irregular,  351 
Malarial  parasites,  349 
asexual  cycle,  349 
blood-films  in,  357 
detection,  354 

by  concentration  methods, 

.  359 
estivo-autumnal,  346 
in  unstained  blood,  355 
life  histories,  349 
mosquitos  as  hosts,  352 
quartan,  349,  350,  356 

older,  detection,  358 


Malarial  parasites,  segmentation 
of,  350,  358 
tertian,  349,  350,  356 

older,  detection,  358 
varieties,  356 
young,  detection,  357 
stippling  in  leukocytes,  319 
Malignant    disease,    leukocytosis 
in,  292 
tumors,  anemia  from,  381 
Maltose  in  urine,  171 
Markers,  object,  for  microscope, 

39 
Marshall's  urease  method  for  ex- 
amining urine,  139 
Mast-cells,  338 

Mastic  test  for  cerebrospinal  fluid, 
528 
preparation  of  solutions, 

529 
technic,  530 
Mastigophora,  452,  460 
Mcjunkin's  device  for  obtaining 

blood,  256 
Measles,  diazo-reaction  in,  189 
Measures,  653 
Meat  adulteration,  precipitin  test 

for,  614 
Media,    culture-,    564.     See   also 

Culture  media. 
Megaloblasts,  321 
-   in  pernicious  anemia,  323 
Megalocytes,  316 
Melanin  in  urine,  183 

tests  for,  184 
Melanuria,  183 
Membrane,  brood,  485 
Meningitis,      epidemic     cerebro- 
spinal, 531 
antimeningococcus-se  rum 
test  for,  530 
Menstruation,  eosinophilia  in,  337 
Mercury  in  urine,  196 
Mesothelial  cells,  predominance, 

523 
Metal,  sterilization  of,  562 
Methemoglobin,  260 

spectroscopic  test  for,  370 
Methylene    blue,    Pappenheim's, 
for  tubercle  bacillus,  641 
stain,  641 
test,  Russo's,  190,  191 


INDEX 


673 


Metric  system,  tables,  653 
Mett's  test  for  gastric  contents, 

413 
Microblasts,  320 
Micrococcus  catarrhalis,  582 

in  sputum,  90 
Microcytes,  316 
MicrofilaricC,  499 
Micrometer,  cardboard,  44 
eye-piece  for  microscope,  42 

step,  43 
lycopodium  as,  44 
Micrometry,  method  of,  44 
Micron,  45 
Microphotography,    camera    for, 

46 
Microscope,  17 
care  of,  40 
carrying,  41 
choice  of,  48 
cleaning,  41 
condenser  for,  25 
cover  for,  41 
curvature  of  field,  29 
electric  lamp  for,  19 
eye-pieces  for,  26 
for  condenser,  20 
for  serum-work,  603 
illumination  for,  18 
amount,  22 
forms,  22 
magnification  by,  34 
empty,  36 

methods  of  increasing,  36 
micrometer  eye-piece  for,  42 
object  markers  for,  39 
objectives  for,  26 
corrections,  27 
pointer  for,  29 
practical  exercises  with,  49 
use  of,  17,  37 
Microscopic  examination  of  bac- 
teria, 577 
of  sputum,  62 
image,  30 

virtual,  30 
objects,  measurement  of,  42 
Micturition,  frequency  of,  102 
Milk,  544 

analysis  of,  544 
tube  for,  546 
bacteria  in,  544 
43 


!Milk,  boric  acid  in,  Goske's  test 
for,  548 
chemical  examination,  544 
fat  in,  546 
formaldehyd  in,  548 
formalin  in,  test  for,  547 
Holt's    apparatus    for   testing, 

545    . 
lactose  in,  estimation  of,  547 
litmus,  preparation  pf,  568 
preservatives  in,  detection,  547 
proteins  in,  estimation,  547 
reaction,  544 
Milk-sugar  in  urine,  171 
Mineral  sulphates  in  urine,  131 
Minot's  modification  of  Moss'  test 
for  agglutination  of  blood,  377 
Mold  fungi  in  urine,  240 
Molds,  hyphae  of,  in  urine,  227 

in  sputum,  73 
Moller's  stain  for  spores,  574 
Mononuclear    leukocytes,    large, 

329 
Morax-Axenfeld,  diplococcus  of, 

540 
Morner's  reagent,  208 

test  for  tyrosin,  208 
Moro  tuberculin  reaction,  597 
Morphin  in  urine,  197 
Morphology,  staining  for,  571 
Mosquitos  as  hosts  for  malarial 

parasites,  352 
Moss'  test,  Minot's  modification, 

for  agglutination  of  blood,  377 
^lotor  power  of  stomach,  420 
Mouth,  diseases  of,  535 

organisms  of,  535 

tuberculosis  of,  540 
Much  granules  in  sputum,  82 

in  sputum  staining  methods 
for,  83 
Mucin  in  urine,  158 
iMucous  threads  in  urine,  225 
Mucus,  Dittrich's  plugs  in,  61 

in  feces,  426 

in  stomach  contents,  399 
Muller,  blood-dust  of,  250 
Muscle-fibers  in  feces,  438 
Myelin  globules  in  sputum,  71 
Myeloblasts,  341 

indophenol    oxydase    test   for, 
342 


674 


INDEX 


Myelocytes,  339 
in  myelogenous  leukemia,  341 
in  pernicious  anemia,  341 
Myelogenous  leukaemia,  387 
acute,  388 
myelocytes  in,  341 
eosinophilia  in,  337 
Myiasis,  513 

Nagana,  tiypanosome  of,  465 
Necator  americanus,  501,  502 
Negri  bodies  in  rabies,  55.5 

Frothingham's  method  for, 

555 

Neisser's     stain    for    diphtheria 
bacillus,  538 

Nemathelminthes,  472,  492 

Nematoda,  492,  494 

Nematodes,  494 

Nephritis,  urine  in,  244,  245 

Nessler's  reagent,  140 

Neubauer    ruling    for    count    in 
leukemia,  295 

Neuroses,   gastric,  stomach  con- 
tents in,  417 

Neutrophils,  Arneth's  classifica- 
tion, 334 

Neutrophilic     leukocytes,     poly- 
morphonuclear, 330 
structures  of  blood,  307 

Nitrogen  equilibrium,  135 
in  urine,  134 
partition,  135 

Noguchi's   butyric   acid   test  for 
globulin,     in     cerebrospinal 
fluid,  525        . 
cutaneous  reaction  for  syphilis, 
598 

Normoblasts,  320 

Nose,  cylindric  cells  from,  in  spu- 
tum, 93 

Nubecula  in  urine,  104 

Nuclear  particles  of  erythrocytes, 
321 

Nucleated    erythrocytes,    signifi-- 
cance  of,  322 

Nuclei  test,  Schmidt's,  for  exami- 
nation of  feces,  446 

Numeric  aperture,  30 

Nummular  sputum,  60 

Nutrition,    poor,    anemia    from, 
381 


Obermayer's  reagent,  134 

test  for  indican  in  urine,  134 
Object  markers  for  microscope,  39 
Objectives,  achrorriatic,  26 

apochromatic,  26 

depth  of  focus,  34 

dry,  28 

for  microscope,  26 
corrections,  27 

immersion,  28 

numeric  aperture,  30 

oil-immersion,  28 

resolving  power  of,  32 

working  distance,  29 
Oblique   illumination   for   micro- 
scope, 22 
Occult   hemorrhage   in  feces,  de- 
tection, 429 
Odor  of  feces,  425 
Oflice  laboratory  equipment,  643 

routine,  643-645 
Oil-immersion  objective,  28 
Oligochromemia,  263 
Oligocythemia,  271 
Oliguria,  102 
Oncospheres,  481 
Ophthalmia,  gonorrheal,  542 
Ophthal mo- tuberculin   reaction, 

Calmette's,  596 
Opisthorchis,  475 

felineus,  475 

sinensis,  475 
Oppenheim  and  Sachs  stain  for 

Treponema  pallidum,  553 
Opsonic  index,  615 

Leishman's  method,  616 
Wright's  method,  615 
Opsonins,  602,  615 
Orcinol  test  for  pentose,  172 
Organic  acids  in  gastric  contents, 
401,  412 

constituents  of  urine,  99,  124 
Oriental  sore,  Leishmania  tropica 

of,  466 
Orthostatic  albuminuria,  150 
Otitis  media,  543 

bacteria  of,  543 
Ova  in  feces,  443 
Oxybutyric  acid  in  urine,  178 
Oxydase   test  for  blood-cells,  342 
Oxyhemoglobin,  260 

spectroscopic  test  for,  370 


INDEX 


675 


Oxyphilic  structures  of  blood,  307 
Oxyuris,  497 

vermicularis,  497 

Pancreatic  ferments  in  feces,  434 
insufficiency,  amylase  test  for, 

148,  435 
trypsin  test  for,  436 
Pandy's  test  for  globulin  in  cere- 
brospinal fluid,  526 
Panoptic  stain  for  blood,  313 
Pappenheim's  method  for  Bacil- 
lus tuberculosis  in  sputum,  78 
methj'lene    blue    for    bacteria, 

641 
panoptic  stain  for  blood,  313 
pyronin-methyl-green  stain  for 

bacteria,  642 
pyronin-methyl-green  stain  for 
blood,  314 
Paragonimus,  476 
kellicotti,  477 
ringeri,  477^ 
westermanii,  477 
Paramoecium  coli,  471 
Parasites,  animal,  448 
anemia  from,  382 
classification,  450 
definitive  host,  449 
in  blood,  348 
in  feces,  427,  443 
in  sputum,  74 
in  urine,  237 
intermediate  host,  449 
blood,  345 
incidental,  472 

malarial,   in   blood,    349.     See 
also  Malarial  parasites. 
Parasitic  diseases  of  skin,  543 
Paratyphoid  fever,  Widal  reaction 
in,  604.     See  also  Widal  reac- 
tion. 
Paroxysmal  hemoglobinuria,  i8i 
Partition,  nitrogen,  135 
Pathologic  polymorph onuclear 

leukocytosis,  290 
Pavement  cells  in  urine,  229 
Pediculus  capitis,  511 

vestimenli,  511 
Pentoses  in  urine,  172 

Bial's  orcinol  test,  17 
Pentosuria,  172 


Pepper's  method  of  concentration 

of  ova  of  hookworm,  505 
Pepsin  in  gastric  contents,  403, 
412 
Hammersclilag's    method, 

412 
Mett's  test,  413 
test  for,  404 
Pepsinogen   in   gastric   contents, 

403 
test  for,  404 
Peptid -split  ting    enzyme  in  gas- 
tric contents,  405 
Peptone      solution,      Dunham's, 

preparation  of,  569 
Pericardial    fluids,    examination, 

520 
Peritoneal  fluid,  examination,  520 
Pernicious     anemia,     383.      See 

also  Anemia,  pernicious. 
Persistent  glycosuria,  161 
Pertussis,    lymphocytic   leukocy- 
tosis in,  293,  327 
Pessary    forms    of    erythrocytes, 

316 
Phagocytic  index,  616 
Phagocytosis,  290 
Pharyngomycosis  leptothrica,  535 
Pharynx,  tuberculosis  of,  540 
Phenacetin  in  urine,  192 
Phenol  in  urine,  197 
Phenolphthalien  in  urine,  197 
Phenolsulphonephthalein  test  for 

urine,  112 
-Phenylhydrazin  test  for  glucose, 

164 
Phosphates  in  urine,  211 
alkaline,  129 
amorphous,  129 
decreased,  130 
earthy,  129 

quantitative  estimation,  131 
triple,  129 
Photomicrography,  45 
Phthirius  pubis,  511 
•Physiologic  albuminuria,  150 
polymorphonuclear       leukocy- 
tosis, 290 
Pigmented  cells  in  sputum,  70 
Pigments  in  urine,  loi 
Pink-eye,  540 
Pin- worm,  497 


676 


INDEX 


Pipets,  561 

for  serum-work,  604 

for  urine,  199 
Piroplasma  hominis,  471 
Plasmodium,  470 

falciparum,'  349 

malariae.     See   Malarial   para- 
sites. 

vivax,  349 
Platinum  wires,  560 
Platyhelminthes,  472,  473 
Plerocercoids,  490 
Pleural  fluid,  examination,  520 
Plugs,  Dittrich's  in  mucus,  61 
Pneumococci,  517 
Pneumococcus,  581 

capsules,  Buerger's  method  for, 

87 
Rosenow's  method  for,  88 
Smith's  method  for,  87 

in  eye  affections,  438 

in  sputum,  85 
Pneumonia,  croupous,  sputum  in, 
60,  97 

drunkard's,  sputum  in,  60 
Poikilocytes,  317 
Poikilocytosis,  317 
Pointer  for  microscope,  29 
Poisoning,  chronic,  anemia  from, 

381 
Polychromatophilia,  317 
Polychrome  methylene-blue-eosin 

stains  for  blood,  309,  312 
Polycythemia,  270 
Polyhedral  cells  in  urine,  227 
Polymorphonuclear    leukocytes, 
predominance,  521 

leukocytosis,  289 

neutrophilic  leukocytosis,  330 
Polynuclear  leukocytes,  330 
Polyuria,  102 
Ponder's     stain     for     diphtheria 

bacillus,  538 
Pork  tapeworm,  484 
Posthemorrhagic  anemia,  382 

leukocytosis,  292 
Postural  albuminuria,  150 
Potassium    indoxyl    sulphate    in 

urine,  132.     See  also  Ittdican  in 

urine. 
Potato  medium,  preparation  of, 

S68 


Power  of  resistance  of  patient,  332 

resolving,  of  objective,  32 
Precipitin  test  for  meat  adultera- 
tion, 614 
for  unknown  proteins,  611 
Precipitins,  601 

Preformed  sulphates  in  urine,  131 
Premyelocytes,  340 
Preservation  of  urine,  loi 
Preservatives  in  milk,  detection 

of,  547 
Primary  anemia,  383 
Proglottides,  480 
Progressive    pernicious    anemia, 

383 
Protein,    Bence-Jones,    in    urine, 
.    ^S8 

in  milk,  estimation,  547 
in  urine,  149 

unknown,    biologic    identifica- 
tion, 611 
Uhlenhuth's  test  for,  611 
Proteoses  in  urine,  160 
Protozoa,  451 
outline,  452 
Prune- juice  sputum,  60 
Pseudocasts  in  urine,  226 
Pseudomembranous       conjuncti- 
vitis, 542 
inflammations,  acute,  537 
Pulmonary  edema,  sputum  in,  96 

tuberculosis,  sputum  in,  97 
Purdy's  centrifugal  estimation  of 
albumin  in  urine,  158 
of  phosphates,  131 
of  sulphates,  131 
centrifuge  tubes,  122 
electric    centrifuge    for    urine, 

121,  122 
heat  test  for  albumin,  156 
table  for  estimation  of  albumin, 

159 
of  chlorids,  1 28 
of  phosphates,  130 
of  sulphates,  132 
Purin  bodies  in  urine,  142 

estimation.  Cook's  method, 

143 
Hall's  method,  143 
quantitative,  142 
Purpura     haemorrhagica     blood- 
plaques  in,  301 


INDEX 


677 


Pus,  examination  of,  515 

gonococci  in,  519 

in  feces,  440 

in  urine,  105 

staphylococci  in,  516 

streptococci  in,  516 
Pus-casts  in  urine,  223 
Pus-cells  in  gastric  contents,  415 
Pus-corpuscles,  331,  515 

in  sputum,  91 

in  urine,  230 

predominance,  521 
Pyelitis,  urine  in,  246 
Pyorrhea   alveolaris,   Endamoeba 

gingivalis  in,  458 
Pyronin  stain,  642 
Pyronin-methyl    green    stain    for 
blood,  314 
Pappenheim's,   for   bacteria, 
642 
Pyuria,  230 

Quartan  malarial  parasites,  349, 
350,  356 
older,  detection,  358 
Quinin  in  urine,  197 

Rabies,  555 
diagnosis,  555 
Negri  bodies  in,  555 

Frothingham's  method  for, 

555 
Racks  for  test-tubes  for  serum- 
work,  603 
Ray-fungus  in  sputum,  72 
Reaction,  diazo,  159 
substitutes  for,  190 
technic,  189 
Wassermann,     619.     See     also 

Wassermann  reaction. 
Widal,    604.     See    also    Widal 
reaction. 
Reagent,  Boas',  401 

Esbach's,  for  albuminuria,  157 
Morner's,  208 
Nessler's,  140 
Obermayer's,  134 
Ruhemann's,  145 
Reagents,  650-652 

preservation  of,  650 
Receptacle  for  sputum,  57 
Receptors  of  first  order,  601 


Receptors  of  second  order,  601 

of  third  order,  602 
Red  blood-corpuscles.     See  Ery- 
throcytes. 

sand  in  urine,  203 

sputum,  59 
Rehfuss  stomach-tube,  396 
Reinsch's  test  for  arsenid'in  ur^ne,--'' 

192 
Relapsing   fever,   spirochetes   of, 

460 
Renal  albuminuria,  150 

calculus,  urine  in,  244 

hyperemia,  urine  in,  242 

tuberculosis,  urine  in,  244 
Rennin  in  gastric  contents,  405 

test  for,  405 
Renninogen  in  gastric  contents, 

495 
Resinous  drugs  in  urine,  198 
Resistance,    patient's    power    of, 

332  _      • 
Resolving  power  of  objective,   32 
Rhabditiform  larvae,  507 
Rhizopoda,  452,  453 
Riegel's  test-meal,  395 
Rimini-Burnam  test  for  formalde- 

hyd  in  urine,  194 
Ring  bodies,  Cabot's,  323 

tests  in  albuminuria,  152 
Ringworm,  543 

Roberts'  differential  density  test 
for  glucose  in  urine,  1 70 

test  for  albuminuria,  154 
Ronchese-Malfatti  method  of  es- 
timating ammonia  in  urine,  147 
Rosenow's  method  for  pneumo- 

coccus  capsules,  88   . 
Rot,  liver,  475 

Rothera's  test  for  acetone,  177 
Rouleaux  formation  of  erythro- 
cytes, 249 
Round  worms,  492,  494,  495 
Rowntree  and  Geraghty's  phenol- 

sulphonephthalein      test      for 

urine,  112 
Ruhemann's  method  of  estimat- 
ing uric  acid  in  urine,  144 

reagent,  145 

uricometer,  145 
Russo's  methylene-blue  test,  191 
Rusty  sputum,  60 


678 


INDEX 


Saccharimeter,  Einhorn's,  168 
Sahli's   desmoid    test   of   gastric 
digestion,  421 

glutoid  test  for  examination  of 
feces,  446 

hemoglobinometer,  266,  267 
Salicylates  in  urine,  198 
Salol  in  urine,  198 

test,  Ewald's,  for  gastric  motil- 
ity, 420 
Sand,  red,  in  urine,  203 
Sarcinae  in  gastric  contents,  415 
Sarcodina,  452,  453 
Sarcoptes  scabiei,  511 
Saxe's  urinopyknometer,  109,  no 
Scales  for  serum-work,  603 
Scarlet  fever,  eosinophilia  in,  337 
Scharlach  R  stain  for  fat,  643 
Schick  test  in  diphtheria.  599 
Schistosomum,  477 

haematobium, '477 

as  cause  of  hemorrhage  from 

bladder,  234 
in  urine,  237 

japonicum,  480 

mansoni,  479 
Schlesinger's  test  for  urobilin,  186 
Schmidt's  diet  for  examination  of 
feces,  444 

nuclei  test  for  examination  of 
feces,  446 

test  for  urobilin  in  feces,  431 
Schiiffner's  granules,  319 
Schwartz  and  McNeil's  reaction 

for  gonorrhea,  632 
Scolex,  480,  481 
Scratches   on   slide   as   cause   of 

error,  241 
Screw  worm,  513 
Sediment,  urinary,  198,     See  also 

Urinary  sediment. 
Segmentation   of   malarial  para- 
sites, 350 
Semen,  553 

Boston's  method  of  transport- 
ing, 553 

examination  of,  553 

on  clothes,  detection  of,  553 
Florence's  reaction  for,  554 
Serodiagnostic  methods,  600 
Serosomucin,  520 
Serum-albumin  in  urine,  149 


Serum-globulin  in  urine,  149 
Serum-water  media.  Hiss',  569 
Sexual  cycle  of  malarial  parasites, 

351 
Shadow  cells  in  urine,  233 
Sheep's   erythrocytes  in  Wasser- 

mann  reaction,  620 
Shellac  cement,  39 
Side-chain   theory  of  immunity, 

Ehrlich's,  600 
Silver   nitrate   solution,   ammon- 
iated,  143,  144 
stain  for  Treponema  pallidum, 
552 
Simon's  tesL  for  lactic  acid,  403 
Skin  diseases,  eosinophilia  in,  337 
parasitic  diseases  of,  543 
test  for  syphilis,  598     . 
Sleeping  sickness,  349 

trypanosome  of,  464 
Smith's  method  for  pneumococ- 
cus  capsules,  87 
test  for  bile  in  urine,  179 
Soaps  in  feces,  440 
Solids,  total,  in  urine,  no 

in  urine,  estimation  of,  in 
Sore,  Oriental,  Leishmania  tropica 

of,  466 
Specific  gravity  of  urine,  107 
Spectroscope,  direct-vision,  366 
Spectroscopic  test  for  blood,  366 
for   carbon    monoxid   hemo- 
globin, 372 
for  hematoporphyrin,  371 
for  hemochromogen,  371 
for  hemoglobin,  183,  371 
for  methemoglobin,  370 
for  oxyhemoglobin,  370 
for  reduced  hemoglobin,  370 
treatment  of  material  for,  368 
Spermatozoa  in  urine,  234 
Spinal    fluid.     See    Cerebrospinal 

fluid. 
Spirals,    Curschmann's,    in    spu- 
tum, 67 
Spirochaeta,  460 
buccalis,  462 
carteri,  461 
dentium,  462 
duttoni,  461 

icterohemorrhagicse,  463 
kochi,  461 


INDEX 


679 


Spirochseta  novyi,  461 

pallida,  548 

recurrentis,  460 

refringens,  462 

vincenti,  462 
Spirochetosis,  bronchial,  463 
Splenic  anemia,  386 
Splenomegaly,    infantile,    Lcish- 

mania  infantum  of,  466 
Spores,  Huntoon's  stain  for    575 

Holler's  stain  for,  574 
Sporozoa,  453,  470 
Spring-lancet,  254 
Sputum,  Actinomyces  bovis  in,  72 

albumin  in,  q4 

animal  parasites  in,  74 

Bacillus  mucosus  capsulatus  in, 
88 
of  Friedlander  in,  88 
of  influenza  in,  89 
pertussis  in,  90 
tuberculosis  in,  76 

bacteria  in,  75 

black,  60 

blood  in,  59 

bronchial  casts  in,  61 

brown,  60 

carbon-laden  cells  in,  71 

cells  in,  90 

chemic  examination,  94 

coin-like  disks  in,  98 

collecting  sample,  56 

color  of,  59 

consistence,  60 

cotton  fibrils  in,  66 

crudum,  60 

crystals  in,  69 

Curschmann's  spirals  in,  67 

cylindric  cells  in,  93 

disposal  of,  58 

elastic  fibers  in,  64 

Endamoeba  histolytica  in,  74 

eosinophilic  cells  in,  91 

epithelial  cells  in,  92 

erythrocytes  in,  94 

examination,  56 

oUecting  sample  for,  56 
macroscopic,  58 
microscopic,  62 
physical,  59 
routine,  57 

Frankel's  diplococcus  in,  85 


Sputum,  globular,  98 

gray,  60 

heart-failure  cells  in,  70 

in  acute  bronchitis,  95 

in  anthracosis,  71 

in  bronchial  asthma,  97 

in  bronchiectasis,  96 

in  chronic  bronchitis,  95 

in  croupous  pneumonia,  60,  97 

in  disease,  95 

in  drunkard's  pneumonia,  60 

in  gangrene  of  lung,  96 

in  pulmonary  edema,  96 
tuberculosis,  97 

larvaj  in,  74 

Leptothrix  buccalis  in,  66 

macroscopic  examination,  59 

micrococcus  catarrhalis  in,  90 

microscopic  examination,  62 

molds  in,  73 

morning,  95 

Much  granules  in,  82 

staining  methods  for,  83 

myelin  globules  in,  71 

nummular,  60 

physical  examination,  59 

pigmented  cells  in,  70 

pneumococcus  in,  85 

prune-juice,  60 

pus-corpuscles  in,  91 

quantity,  59 

ray-fungus  in,  72 

receptacle  for,  57 

red,  59 

routine  examination,  57 

rusty,  60 

squamous  cells  in,  92 

stained,  74 

staphylococcus  in,  85 

streptococcus  in,  85 

Streptothrix  actinomyces  in,  72 

Strongyloides  intestinalis  in,  74 

Trichomonas  intestinalis  in,  74 

tubercle  bacillus  in,  76 

unstained,  63 

yeasts  in,  73 

yellowish-green,  59 
Squamous  cells  in  sputum,  92 

in  urine,  229 
Squibb's  urinometer,  108 
Stained  sputum,  74 
Staining  for  morphology,  571 


68o 


INDEX 


Staining  methods,  571 

solutions,  639 
Stains,  639,  652 

anilin-gentian  violet,   for   bac- 
teria, 640 
Buerger's,    for    pneumococcus 

capsules,  87 
carbol-methyl  violet,  84 
carbol-thionin  for  bacteria,  639 

for  blood,  314 
Czaplewski's  carbol-fuchsin,  for 
bacteria,  640 
carbol-gentian  violet,  for  bac- 
teria, 640 
for  blood-films,  302,  307 
formalin-gentian  violet,  for 

bacteria,  640 
fuchsin,  for  Bacillus  tuberculo- 
sis, 639 
Gabbet's,  for  tubercle  bacilli  in 
sputum,  78 
for  tubercle  bacillus,  641 
gentian  violet,  for  bacteria,  640 
Giemsa's,  for  blood,  313 

for  Treponema  pallidum,  550 
Gram's,  for  bacteria,  572 
Harris'   hematoxylin,   for  bac- 
teria, 640 
hematoxylin,  for  bacteria,  640 
Huntoon's,  for  spores,  575 
India-ink,  for  Treponema  palli- 
dum, 552 
iodine,  for  bacteria,  641 
Jenner's,  for  blood,  313 
LofBer's     alkaline     methylene 

blue,  for  bacteria,  641 
Loffler's,  for  flagella,  575 
methylene  blue,  641 
Moller's,  for  spores,  574 
Neisser's,  for  diphtheria  bacil- 
lus, 538 
Oppenheim     and     Sachs,     for 

Treponema  pallidum,  553 
Pappenheim's      for      tubercle 
bacilli  in  sputum,  78 
methylene  blue,  for  tubercle 

bacillus,  641 
panoptic,  for  blood,  313 
pyronin- methyl     green,     for 
blood,  314 
polychrome  methylene  -  b  1  u  e  - 
eosin,  for  blood,  309 


Stains,    Ponder's,    for   diphtheria 

bacillus,  538 
preservation  of,  649 
pyronin,  642 
Rosenow's,   for   pneumococcus 

capsules,  88 
Schariach  R,  for  fat,  643 
silver,  for  Treponema  pallidum, 

simple  bacterial,  642 
Smith's,  for  pneumococcus  cap- 
sules, 87 
Sudan  HI,  for  fat,  643 

Herxheimer's,  643 
Wright's,  for  blood,  309 
application,  310 
preparation,  310 
Ziehl-Neelson,  for  tubercle  bac- 
illi in  sputum,  78 
Staphylococcus  in  eye  affections, 
.438  • 

in  pus,  516 
in  sputum,  85 
pyogenes  albus,  581 
aureus,  580 
citreus,  581 
Starch-granules  in  feces,  438 
Steam  sterilizer,  559 
Step  micrometer  eye-piece,  43 
Sterilization,  562 
of  cotton,  563 
of  culture-media,  562 
of  gauze,  563 
of  glassware,  562 
of  metal,  562 
of  vaccines,  588 
Sterilizers,  558 
dry,  558 

hot-air,  558  ^        f 

steam,  559  f    /     ,    ^ 

'"Stippling,  basophilic,  3i8(^"  \> 

malarial,  in  leukocytes,  319 
Stock  vaccines,  585 
Stomach,  319 

absorptive  power  of,  419 
contents  of,  392.     See  also  Gas- 
tric contents. 
digestion,  392 

Sahli's     desmoid      test     of, 
421 
dilatation  of,  gastric  contents 
in,  417 


INDEX 


681 


Stomach,  diseases  of,  indican  in 
urine  in,  133 

motor  power  of,  420 

position  of,  determination,  421 

size  of,  determination,  421 

worm,  496 
Stomach-tube,    introduction    of, 

395 
Rehfuss,  396 
Stools,  423.     See  also  Feces. 
Storage  of  culture-media,  571 
Strauss'  test  for  lactic  acid,  403 
Streptococcus   in   eye  affections, 
438 
in  pus,  516 
.  in  sputum,  85 
pyogenes,  581 
viridans,  581 
Streptothrix  actinomyces  in  spu- 
tum, 72 
Strongyloides,  506 
intestinalis,  506 
in  sputum,  74 
Sudan  III,  stain  for  fat,  643 

Herxheimer's,  643 
Sugar,  fruit,  in  urine,  170 
in  cerebrospinal  fluid,  530 
media,  preparation  of,  567 
Sugars  in  urine,  160 
Sulphates  in  urine,  131 
conjugate,  132 

estimation,  Purdy's  centrifu- 
gal method,  131 
Purdy's   table,   after   cen- 

trifugation,  132 
quantitative,  131 
ethereal,  132 
mineral,  131 
preformed,  131 
Sulphosalicylic  acid  test  for  albu- 
minuria, 154 
Suppression  of  urine,  103 
Surra,  trypanosome  of,  465 
Suspension,  making  of,  for  vac- 
cines, 587 
Syphilis,    cobra-venom    test    for, 
636 
materials  required,  636 
technic,  637 
complement  deviation  test  for, 

619 
cutaneous  test  for,  598 


Syphilis,    dark-ground    illumina- 
tion for,  552 
examination  of  material,  548 
Giemsa's  stain  for,  550 
hereditary,  lymphocytic  leuko- 
cytosis in,  293 
India-ink  method  for,  552 
Oppenheim    and    Sachs'    stain 

.  for,  553 

silver  stain  for,  552 
Wassermann  reaction  in,  619. 
See   also    Wassermann   reac- 
tion. 
Wright's  blood-stain  for,  551 
Syphilitic    antigen    in,    Wasser- 
mann reaction,  619 
Syringe,  Luer  all-glass,  for  serum- 
work,  604 

T^NIA,  482 

echinococcus,  481,  485 
in  urine,  237 

elliptica,  489 

saginata,  480,  482 

solium,  480,  481,  484 
Tallqvist's  hemoglobin  scale,  269 
Tannin  in  urine,  198 
Tapeworm,  473,  480 

beef,  482 

dwarf,  487 

fish,  490 

pork,  484 
Teichmann's  test  for  blood,  365 
Telosporidia,  453,  470 
Temperature,    table    of    equiva- 
lents, 654 
Tertian   malarial  parasites,   349, 
350,  356 
older,  detection,  358 
Test-breakfast,  Boas',  394 

Ewald's,  394 
Test-meals,  394 

Fischer's,  395 

Riegel's,  395 
Test-tubes  for  serum-work,  603 
Textile  fibers  in  urine,  241 
Thoma-Metz  hemacytometer,  283 
Thoma-Zeiss  hemacytometer,  273 
cleaning  instrument,  280 
sources  of  error,  279 
technic,  273 
Thorn-apple  crystals  in  urine,  215 


682 


INDEX 


Threads,    gonorrheal,    in     urine, 

237,  519 
Thread-worm,  497,  498 
Thrombin,  257 
Thrush,  537 

fungus,  537 
Tinea  versicolor,  543 
Tissue,  bits,  in  gastric  contents, 

399 
Toisson's   fluid  for   blood-count, 

279 
Topfer's  test  for  combined  hydro- 
chloric acid,  411 
for  free  hydrochloric  acid,  410 
for   total   acidity   of   gastric 
contents,  408 
T.  O.  tuberculin,  595 
Toxic  absorption,  degree  of,  331 
Toxocara  canis,  496 
Trachea,  cylindric  cells  from,  in 

sputum,  93 
Trachoma  bodies,  542 
Transfusion,   bloods  for,  match- 
ing, 375 
of  blood,  grouping  individuals 
for,  376,  379 
Transient  leukocytosis,  288 
Transitional  leukocytes,  329 
Transitory  glycosuria,  161 
Transudates,  520 
Trematoda,  473,  474 
Treponema,  463 

pallidum,  463,  548,  549' 
Giemsa's  stain  for,  550 
India-ink  method,  552 
silver  stain  for,  552 
Wright's  blood-stain  for,  551 
pertenue,  463 
Trichinella,  508 
spiralis,  508 

larvae  in  blood,  363 
Trichiniasis,  509 
diagnosis,  509 
eosinophilia  in,  337 
Trichloracetic  acid  test  for  albu- 
minuria, 153 
Trichocephalus,  510 

trichiuris,  510 
Trichomonas,  467 
intestinalis,  467 
in  sputum,  74 
in  urine,  237 


Trichomonas  pulmonalis,  468 

vaginalis,  468 
Triple  phosphates  in  urine,  1 29 
Tropical    dysentery,   Endamoeba 

histolytica  in,  453 
Trypanosoma,  464 

brucei,  465 

cruzi,  465 

equiperdum,  465 

evansi,  465 

gambiense,  464 

lewisi,  465 

rhodesiense,  465 

Trypsin  in  feces,  434 

test  for,  436 

test  for  pancreatic  insufliciency, 
436 
T.  R.  tuberculin,  595 
Tsuchiya's  method  of  estimating 

albumin  in  urine,  157 
Tube-casts  in  urine,  216 

examination  for,  218 
Tube-length,  measurement,  27 
Tubercle   bacillus.     See   Bacillus 

tuberculosis. 
Tuberculins,  594 

action  of,  595 

B.  E.,  595 

B.  F.,  595 

dosage,  595 

in  diagnosis,  596 

Calmette's    ophthalmo-reac- 

tion,  596 
hypodermic  injection,  596 
Moro  reaction,  597 
Von  Pirquet's  reaction,  597 

in  tuberculosis,  594 

reactions  from,  595 

T.  O.,  595 

T.  R.,  595 

varieties,  595 
Tuberculosis,  bacillus  of,  584 

blood-plaques  in,  301 

complement  deviation  test  for, 

633 
diazo  reaction  in,  189 
of  mouth,  540 
of  pharynx,  540 
renal,  urine  in,  244 
sputum  in,  97 
tuberculin  in,  594 
vaccines  in,  594 


INDEX 


683 


Tubing  culture-media,  571 
Tumors,  malignant,  anemia  from, 
381. 
urine  in,  247 
Turck's  irritation  leukocytes,  343 
ruling  for  blood-count  in  leu- 
kemia, 294 
Two-slide  method  for  blood-films, 

303 
Typhoid  bacillus,  583 
in  blood,  346 
technic,  346 
diagnosticum  of  Ficker,  614 
fever,  diazo  reaction  in,  188 
prophylactic  vaccines  in,  594 
Widal  reaction  in,  604.     See 
also  Widal  reaction. 
Tyrosin  crystals  in  urine,  208 
in  urine,  207 


Uffelmann's  test  for  lactic  acid, 

402 
Uhlenhuth's    test    for    unknown 

proteins,  611 
Ulcer,  gastric,  gastric  contents  in, 

419 
Ulrich's  test  for  albuminuria,  155 
Uncinariasis,  eosinophilia  in,  337 
Unstained  sputum,  63 
Urates,  amorphous,  in  urine,  105, 
142,  20s 
in  mass,  226 
Urea  in  urine,  134 
decreased,  136 

estimation,  Folin  and  Denis' 
method,  140 
hypobromite  method,  137 
Marshall's  method,  139 
quantitative,  137 
urease  methods,  139 
increased,  135 
retention,  136 
Urease    methods    of    estimating 

urea  in  urine,  139 
Ureometer,  Doremus-Hinds',  137 
Uric  acid  in  urine,  142 
endogenous,  142 
estimation,  Cook's  method, 

143 
Hall's  method,  143 
quantitative,  144,  146 


Uric  acid   in   urine,   estimation, 
Ruhemann's       method, 
144 
exogenous,  142 
Uric-acid  crystals  in  urine,  203 
Uricometer,  Ruhemann's,  145 
Urinary  sediment,  198 
examination  of,  199 
identification  table,  203 
organized,  215 
unorganized,  201 
in  acid  urine,  202 
in  alkaline  urine,  202,  211 
Urine,  99 

abnormal  constituents,  149 
acetanilid  in,  192 
aceto-acetic  acid  in,  177 
acetone     in,     173.     See     also 
A  cetonuria. 
bodies  in,  172 
acid  fermentation,  106 

unorganized     sediment     in, 
202,  203 
acidity  of,  estimation  of,  Folin's 

method,  107 
air-bubbles  in,  241 
albumin  in,  149.     See  also  Al- 

buminuria. 
alkaline,  unorganized  sediment 

in,  202,  211 
alkalinity  of,  fixed,  107 

volatile,  107 
alkapton  bodies  in,  183 
ammonia  in,  145 
ammoniacal  decomposition  of, 

106 
ammonio-magnesiu  m      p  h  o  s  - 
phate  in,  211 
crystals  in,  211 
ammonium   urate   crystals   in, 

215 
amorphous  phosphates  in,  214 

urates  in,  205 
in  mass,  226 
amylase  in,  148 
Anguillula  aceti  in,  237 
animal  parasites  in,  237 
antipyrin  in,  192 
arsenic  in,  192 

Gutzeit's  test  for,  192 

Reinsch's  test  for,  192 
aspirin  in,  198 


684 


INDEX 


Urine,  atropin  in,  193 

Bacillus  tuberculosis  in,  235 

detection  of,  236 
bacteria  in,  105,  234 
bacterial  casts  in,  224 
Bence- Jones  protein  in,  158 
bile  in,  178 

Gmelin's  test  for,  180 

Smith's  test  for,  180 
bile-acids  in,  detection,  180 

Hay's  test  for,  180 
bile-pigment  in,   detection   of, 

179 
blood  in,   105,   223,   232,   233. 

See  also  Hematuria. 
brick-dust  deposit  in,  105 
bromids  in,  193 
calcium  carbonate  in,  214 

oxalate  in,  205 
cane-sugar  in,  171 
casts  in,  containing  organized 
structures,  223 

structures  simulating,  225 
Charcot-Leyden     crystals     in, 

69. 

chemic  examination,  116 

centrifugal  methods,  120 
colorimetric  methods,  116 

chloral  hydrate  in,  193 

chlorids     in,     124.     See     also 
Chlorids  in  urine. 

chyle  in,  211 

clarification  of,  loi 

collection  of  specimen,  100 

color,  103 

cylindroids  in,  226 

cystin  crystals  in,  208 

decomposition  of,  100 

decrease  of,  102 

dextrose  in,  161.     See  also  Gly- 
cosuria. 

diacetic  acid  in,  177 
detection,  177 
Gerhardt's  test,  177 

diazo  substances,  188 

dicalcium    phosphate    crystals 
in,  213 

drugs  in,  191 

envelope  crystals  in,  205 

epithelial  casts  in,  223 
cells  in,  227 

erythrocytes  in,  232 


Urine,  examination,  chemic,  116 
centrifugal  methods,  120 
colorimetric  methods,  116 
microscopic,  198 
physical,  102 
urease  methods,  139 

Folin  and  Denis,  140 
Marshall's,  139 
extraneous  structures  in,  239 
fat-droplets  in,  241 
fat-globules  in,  210 
fatty  casts  in,  222 
fibers  in,  227 
fibrinous  casts  in,  221 
fixed  alkalinity  of,  107 
floaters  in,  237 
formaldehyd    in,    Rimini-Bur- 

nam  test  for,  194 
fruit  sugar  in,  170 
functional  tests  for,  112 
glucose  in,  i6i.     See  also  Gly- 
cosuria. 
gonococci  in,  237 
gonorrheal  threads  in,  225,  237, 

granular  casts  in,  222 
gravel  in,  203 
hairs  in,  227 

hematoporphyrin  in,  184 
hemoglobin  in,  180.     See  also 

Hemoglobinuria . 
hexamethylenamin  in,  193 
hyaline  casts  in,  219 
hyphae  of  molds  in,  227 
identification  of,  loi 
in  cystitis,  246 
in  diabetes  insipidus,  248 

mellitus,  248 
in  disease,  242 
in  nephritis,  244,  245 
in  pyelitis,  246 
in  renal  calculus,  244 

hyperemia,  242 

tuberculosis,  244 
in  tuberculosis,  247 
in  vesical  calculus,  247 
increase  of,  102 
indican  in,  132.     See  also  Indi- 

can  in  urine. 
inorganic  constituents,  99,  124 
irregular  cells  in,  228 
lactose  in,  171 


INDEX 


68s 


Urine,  lead  in,  194 

Lederer's  test  for,  194 
leucin  in,  207 
levulose  in,  170.     See  also  Levu- 

lose  in  urine. 
lycopodium  granules  in,  241 
maltose  in,  171 
melanin  in,  183 

tests  for,  184 
mercury  in,  196 
microscopic  examination,  198 
milk-sugar  in,  171 
mold  fungi  in,  240 
morphin  in,  197 
mucin  in,  158 
mucous  threads  in,  225 
muscle  fibers  in,  242 
nitrogen  in,  134 
normal  constituents,  123 
nubecula  in,  104 
organic  constituents,  99,  124 
oxybutyric  acid  in,  178 
pavement  cells  in,  229 
pentoses  in,  172 

Bial's  orcinol  test,  172 
phenacetin  in,  192 
phenol  in,  197 
phenolphthalein  in,  197 
phenolsulphonephthalein      test 

for,  112 
phosphates  in,   129,   211.     See 

also  Phosphates  in  urine. 
physical  examination,  102 
pigments  in,  loi 
pipets  for,  199 
polyhedral  cells  in,  227 
potassium  indoxyl  sulphate  in, 

132.     See    also    Indican    in 

urine. 
preservation  of,  loi 
proteins  in,  149 
proteoses  in,  160 
pseudocasts  in,  226 
purine  bodies  in,  142 

estimation.    Cook's    meth- 
od, 143 
Hall's  method,  143 
quantitative    estimation, 
142 
pus  in,  105 
pus-casts  in,  223 
pus-corpuscles  in,  230 


Urine,  quantitative  estimation  of 

acidity,  107 
quantity  passed,  102 
quinin  in,  197 
reaction,  106 
red  sand  in,  203 
resinous  drugs  in,  198 
salicylates  in,  198 
salol  in,  198 
Schistosomum  haematobium  in, 

237 
sediment    in,     198.     See    also 

Urinary  sediment. 
serum-albumin  in,  149 
serum-globulin  in,  149 
shadow  cells  in,  233 
small  crystals  in,  226 
specific  gravity,  107 
spermatozoa  in,  234  ' 

squamous  cells  in,  229 
sugars  in,  160 
sulphates    in,    131.     See    also 

Sulphates  in  urine. 
suppression  of,  103 
Taenia  echinococcus  in,  237 
tannin  in,  198 
textile  fibers  in,  241 
thorn-apple  crystals  in,  215 
total  solids  in,  no 

estimation  of,  in 
transparency  of,  104 
Trichomonas  intestinalis  in,  237 
tube-casts  in,  2x6 

examination  for,  218 
tyrosin  in,  207 

unorganized  sediments  in,  201 
urates  in,  amorphous,  142 
urea  in,  134.     See  also  Urea  in 

urine. 
uric    acid    in,    142.     See    also 

Uric  acid  in  urine. 
uric-acid  crystals  in,  203 
urobilin  in,  184 

quantitative  estimation,  187 

Schlesinger's  test  for,  186 
urobilinogen  in,  185 

Ehrlich's  test  for,  186 

quantitative  estimation,  187 
vinegar  eel  in,  237 
volatile  alkalinity  of,  107 
waxy  casts  in,  221 
yeast-cells  in,  239 


686 


INDEX 


Urinometer,  Squibb's,  io8 
Urinopykno meter,  Saxe's,  109 
Urobilin  in  feces,  430 

detection,  431 

Schmidt's  test,  431 

Wilber  and  Addis'  test,  432 
in  urine,  184 

quantitative  estimation,  187, 

431. 
Schlesinger's  test  for,  186 

Urobilinogen  in  urine,  185 
Ehrlich's  test  for,  186 
quantitative  estimation,  187 

Urochromogen  test,  Weis's,  191 


Vaccinls,  autogenous,  585 
bottles  for,  586 
contra-indications,  593 
counting  of,  589 

hemacytometer  method,  S9q 
dosage,  592 
indications  for,  593 
in  infections,  593 
in  malignant  endocarditis,  593 
in  tuberculosis,  594 
making  suspension  for,  587 
method  of  use,  591 
preparation  of,  585 

diluting,  591 

materials,  585 

obtaining  bacteria,  587 
prophylactic,  in  typhoid  fever, 

594 
sterilization  of,  588 
stock,  585 
therapeutic  indications,  592 

Vacuolated  leukocytes,  343 

Vacuum  tube,   Keidel's,  for  col- 
lecting blood,  256 

Vegetable  cells  in  feces,  428,  438 
fibers  in  feces,  428,  438 
hairs  in  feces,  438 

Vernal  catarrh,  eosinophilic  leu- 
kocytes in,  542 

Vesical  calculus,  urine  in,  247 

Vincent's  angina,  539 
spirochete  of,  462 

Vinegar  eel,  494 
in  urine,  237 

Viscosity  of  blood,  379 

VolatUe  alkalinity  of  urine,  107 


Volhard's  method  for  chlorids  in 

urine,  127 
Volume  index  of  blood,  285 

Larrabee's  method,  286 
Volumetric     pipets     for     serum- 
work,  604 
Von  Fleischi's  estimation  of  hemo- 
globin, 265 
hemoglobinometer,  264 
Von  Jacksch's  anemia,  389 
Von  Pirquet's  tuberculin  reaction, 
597 


Wassermann  reaction,  619 

amboceptor   in,  titration  of, 

624 
antigen  in,  titration  of,  625 
antisheep     amboceptor     for, 

620 
complement  in,  621 

titration  of,  623 
errors  and  their  causes,  626 
interpretation  of  results,  630 
materials  required,  619 
patient's  serum  in,  622 
routine  methods,  631 
setting  up,  627 
sheep's  red  blood-cells  in,  620 
syphilitic  antigen  in,  619 
titrations  in,  623 
with  cerebrospinal  fluid,  628 
Water-bath  for  serum-work,  603 
Water-motor  centrifuge  for  urine, 

121 
Watery  blood,  251 
Waxy  casts  in  urine,  221 
Weights,  653 

Weil's  cobra- venom  test  for  syphi- 
lis, 636 
materials  required,  636 
technic,  637 
Weis's  urochromogen  test,  191 
Whip-worm,  510 

White     corpuscles.     See     Leuko- 
cytes. 
Widal  reaction,  604 

interpretation  of  results,  610 
macroscopic,  607 
materials  required,  605 
microscopic,  608 
obtaining  blood,  606 


INDEX 


687 


Wilber   and    Addis'    method    for 

urobilin  in  feces,  432 
Wires,  platinum,  560 
Worm,  flat,  473 

fluke,  473 

guinea-,  500 

hook-,  501 

pin-,  ^97 

round,  492,  494,  495 

screw,  513 

stomach,  496 

tape-,  473,  480 

thread-,  497,  498 

whip-,  510 
Wright's  blood-stain  for  Trepon- 
ema pallidum,  551 

capsule,  562 

cultural  method  for  anaerobes, 
S8o 


Wright's    method     for    counting 
vaccines,  589 
for  opsonic  index,  615 
stain  for  blood,  309 
application,  310 
preparation,  310 
Wright  and   Kinnicutt's  estima- 
tion of  blood-plaques,  301 

Yaws,   Treponema  pertenue  01, 

463 
Yeast-cells  in  gastric  contents,  415 

in  urine,  239 
Yeasts  in  sputum,  73 

Zappert-Ewing  ruling  for  count 

in  leukemia,  295 
Ziehl-Neelson  method  for  Bacillus 

tuberculosis,  in  sputum,  78 
Zoomastigophora,  452,  460 


-^ 


TESTS  OF  RENAL  FUNCTION 

Abstracted  from  "Manual  of  Medicine" 
By  A.  S.  WOODWARK,  M.D.,  F.R.C.P. 


)f  the  many  tests  in  use  at  pres- 
for  renal  function  the  follow- 
are  the  most  important: 

1 )  The  Urea  Concentration  Test 
^'he  patient,  after  emptying  his 
dder,  takes  15  grams  of  urea 
solved  in  100  c.c.  of  water;  at 
end  of  the  first  and  second  hour 
urea  passed  in  the  urine  is  esti- 

;ed  by  the  hypo-bromite  method. 
less  than  2  per  cent  of  urea  is 
sent  the  kidneys  are  stated  to 
inefficient. 

2)  The  Blood  Urea  Test— The 
:)d  may  reveal  the  presence  of 
stances  which  inefficient  kid- 
s  have  failed  to  excrete.  The 
mal  amount  of  urea  in  the 
)d  ranges  from  .02  per  cent  to 

per  cent  (approaching  the 
er  figure  in  older  persons).  It 
assumed  that  no  other  causes 

present  for  high  blood  urea, 
,  cardiac  failure,  suppuration 
loss  of  fluid  in  such  conditions 
[liarrhea. 

b)  The  Chloride  Test— The  per- 
tage  of  Sodium  Chloride  is  esti- 
;ed  after  the  patient  has  been 

on  a  diet  of  known  chloride 


content  for  some  days.  5  gr 
of  salt  are  then  administered, 
the  output  of  chlorides  is 
mated.  According  to  the  ami 
excreted  the  test  will  indicate 
presence  or  absence  of  glomer 
and  tubular  changes  occurriu] 
nephritis. 

(4)  Diastase  Test— 6  to  20  u 
(some  authorities  say  10  to  30 
Diastase  after  entering  the  b 
from  the  pancreas  is  nornf 
secreted  into  the  urine.  If  r 
permeability  is  diminished  the 
put  falls  to  5  units  or  even  n 
The  quantity  is  determined  by 
amount  of  starch  paste  digestei 
a  given  volume  of  urine. 

(5)  Phenolsulphonephtha 
Test — Having  emptied  the  blad 
a   hypodermic    injection    is   n 
into  the  lumbar  muscles  consis 
of  1  c.c.  sterile  salt  solution 
taining    .006    gram    of   the    d 
The  urine  is  collected  one  and 
hours  later.    By  a  colorimeter 
amount  of  dye  passed  is  estimj 
quantitatively,    and    if    less    i 
normal    (=  50  per  cent)    is 
dence  of  renal  inefficiency. 


r- 


A  000  502  520  o 


T63hc 
Todd.  1919 

Clinical  diagnosis 


QYU 

T63^c 

1919 


Todd. 

Clirical  diagnosis 


CALIFORNIA  COLLEGE  OF  MEDICINE  LIBRARY 

UNIVERSITY  OF  CALIFORNIA.  IRVINE 

IRVINE,  CALIFORNIA  92664 


m 


riuKTia  IM  u.«.A- 


